Co-reporter:Huabing Sun, Marisa L. Taverna Porro, and Marc M. Greenberg
The Journal of Organic Chemistry October 20, 2017 Volume 82(Issue 20) pp:11072-11072
Publication Date(Web):October 10, 2017
DOI:10.1021/acs.joc.7b02017
Thymidine radical cation (1) is produced by ionizing radiation and has been invoked as an intermediate in electron transfer in DNA. Previous studies on its structure and reactivity have utilized thymidine as a precursor, which limits quantitative product analysis because thymidine is readily reformed from 1. In this investigation, radical cation 1 is independently generated via β-heterolysis of a pyrimidine radical generated photochemically from an aryl sulfide. Thymidine is the major product (33%) from 1 at pH 7.2. Diastereomeric mixtures of thymidine glycol and the corresponding 5-hydroxperoxides resulting from water trapping of 1 are formed. Significantly lower yields of products such as 5-formyl-2′-deoxyuridine that are ascribable to deprotonation from the C5-methyl group of 1 are observed. Independent generation of the N3-methyl analogue of 1 (NMe-1) produces considerably higher yields of products derived from water trapping, and these products are formed in much higher yields than those attributable to the C5-methyl group deprotonation in NMe-1. N3-Methyl-thymidine is, however, the major product and is produced in as high as 70% yield when the radical cation is produced in the presence of excess thiol. The effects of exogenous reagents on product distributions are consistent with the formation of diffusively free radical cations (1, NMe-1). This method should be compatible with producing radical cations at defined positions within DNA.
Co-reporter:Rakesh Paul, Samya Banerjee, and Marc M. Greenberg
ACS Chemical Biology June 16, 2017 Volume 12(Issue 6) pp:1576-1576
Publication Date(Web):May 1, 2017
DOI:10.1021/acschembio.7b00259
DNA repair is vital to maintaining genome integrity but thwarts the effects of cytotoxic agents that target nucleic acids. Consequently, repair enzymes are potential targets for molecules that modulate cell function and anticancer therapeutics. DNA polymerase β (Pol β) is an attractive target because it plays a key role in base excision repair (BER), a primary pathway that repairs the effects of many DNA damaging agents. We previously identified an irreversible inhibitor of Pol β whose design was based upon a DNA lesion that inactivates Pol β and its back up BER enzyme, DNA polymerase λ (Pol λ). Using this molecule as a starting point, we characterized an irreversible inhibitor (13) of Pol β (IC50 = 0.4 μM) and Pol λ (IC50 = 0.25 μM) from a 130-member library of candidates that is ∼50-fold more effective against Pol β. Pro-13 (5 μM) is only slightly cytotoxic to human cervical cancer cells (HeLa) but potentiates the cytotoxicity of methyl methanesulfonate (MMS). DNA isolated from HeLa cells treated with MMS contains a ∼3-fold greater amount of abasic sites when pro-13 is present, consistent with inhibition of DNA repair. Proinhibitor pro-13 continues to induce cytotoxicity in DNA damaged cells following MMS removal. HeLa cell cytotoxicity is increased ∼100-fold following an 8 h incubation with pro-13 after cells were originally subjected to conditions under which 20% of the cells survive and reproduce. The potentiation of MMS cytotoxicity by pro-13 is greater than any previously reported BER enzyme repair inhibitor.
Co-reporter:Liwei Zheng, Lu Lin, Ke Qu, Amitava Adhikary, Michael D. Sevilla, and Marc M. Greenberg
Organic Letters December 1, 2017 Volume 19(Issue 23) pp:6444-6444
Publication Date(Web):November 10, 2017
DOI:10.1021/acs.orglett.7b03368
Photochemical precursors that produce dA• and dG(N2–H)• are needed to investigate their reactivity. The synthesis of two 1,1-diphenylhydrazines (1, 2) and their use as photochemical sources of dA• and dG(N2–H)• is presented. Trapping studies indicate production of these radicals with good fidelity, and 1 was incorporated into an oligonucleotide via solid-phase synthesis. Cyclic voltammetric studies show that reduction potentials of 1 and 2 are lower than those of widely used “hole sinks”, e.g., 8-oxodGuo and 7-deazadGuo, to investigate DNA–hole transfer processes. These molecules could be useful (a) as sources of dA• and dG(N2–H)• at specific sites in oligonucleotides and (b) as “hole sinks” for the study of DNA–hole transfer processes.
Co-reporter:Liwei Zheng, Markus Griesser, Derek A. Pratt, and Marc M. Greenberg
The Journal of Organic Chemistry April 7, 2017 Volume 82(Issue 7) pp:3571-3571
Publication Date(Web):March 20, 2017
DOI:10.1021/acs.joc.7b00093
Formal hydrogen atom abstraction from the nitrogen–hydrogen bonds in purine nucleosides produces reactive intermediates that are important in nucleic acid oxidation. Herein we describe an approach for the independent generation of the purine radical resulting from hydrogen atom abstraction from the N6-amine of 2′-deoxyadenosine (dA•). The method involves sequential Norrish Type I photocleavage of a ketone (7b) and β-fragmentation of the initially formed alkyl radical (8b) to form dA• and acetone. The formation of dA• was followed by laser flash photolysis, which yields a transient with λmax ≈ 340 nm and a broader weaker absorption centered at ∼560 nm. This transient grows in at ≥2 × 105 s–1; however, computations and reactivity data suggest that β-fragmentation occurs much faster, implying the consumption of dA• as it is formed. Continuous photolysis of 7b in the presence of ferrous ion or thiophenol produces good yields of dA, whereas less reactive thiols afford lower yields presumably due to a polarity mismatch. This tandem photochemical, β-fragmentation method promises to be useful for site-specific production of dA• in nucleic acid oligomers and/or polymers and also for the production of aminyl radicals, in general.
Co-reporter:Samya Banerjee, Supratim Chakraborty, Marco Paolo Jacinto, Michael D. Paul, Morgan V. Balster, and Marc M. Greenberg
Biochemistry 2017 Volume 56(Issue 1) pp:
Publication Date(Web):December 22, 2016
DOI:10.1021/acs.biochem.6b01144
DNA is rapidly cleaved under mild alkaline conditions at apyrimidinic/apurinic sites, but the half-life is several weeks in phosphate buffer (pH 7.5). However, abasic sites are ∼100-fold more reactive within nucleosome core particles (NCPs). Histone proteins catalyze the strand scission, and at superhelical location 1.5, the histone H4 tail is largely responsible for the accelerated cleavage. The rate constant for strand scission at an abasic site is enhanced further in a nucleosome core particle when it is part of a bistranded lesion containing a proximal strand break. Cleavage of this form results in a highly deleterious double-strand break. This acceleration is dependent upon the position of the abasic lesion in the NCP and its structure. The enhancement in cleavage rate at an apurinic/apyrimidinic site rapidly drops off as the distance between the strand break and abasic site increases and is negligible once the two forms of damage are separated by 7 bp. However, the enhancement of the rate of double-strand break formation increases when the size of the gap is increased from one to two nucleotides. In contrast, the cleavage rate enhancement at 2-deoxyribonolactone within bistranded lesions is more modest, and it is similar in free DNA and nucleosome core particles. We postulate that the enhanced rate of double-strand break formation at bistranded lesions containing apurinic/apyrimidinic sites within nucleosome core particles is a general phenomenon and is due to increased DNA flexibility.
Co-reporter:Lakshmi S. Pidugu, Joshua W. Flowers, Christopher T. Coey, Edwin Pozharski, Marc M. Greenberg, and Alexander C. Drohat
Biochemistry 2016 Volume 55(Issue 45) pp:6205
Publication Date(Web):November 2, 2016
DOI:10.1021/acs.biochem.6b00982
Thymine DNA glycosylase (TDG) is a base excision repair enzyme with key functions in epigenetic regulation. Performing a critical step in a pathway for active DNA demethylation, TDG removes 5-formylcytosine and 5-carboxylcytosine, oxidized derivatives of 5-methylcytosine that are generated by TET (ten–eleven translocation) enzymes. We determined a crystal structure of TDG bound to DNA with a noncleavable (2′-fluoroarabino) analogue of 5-formyldeoxycytidine flipped into its active site, revealing how it recognizes and hydrolytically excises fC. Together with previous structural and biochemical findings, the results illustrate how TDG employs an adaptable active site to excise a broad variety of nucleobases from DNA.
Co-reporter:Rakesh Paul and Marc M. Greenberg
The Journal of Organic Chemistry 2016 Volume 81(Issue 19) pp:9199-9205
Publication Date(Web):September 26, 2016
DOI:10.1021/acs.joc.6b01760
The C2′-carbon–hydrogen bond in ribonucleotides is significantly weaker than other carbohydrate carbon–hydrogen bonds in RNA or DNA. Independent generation of the C2′-uridine radical (1) in RNA oligonucleotides via Norrish type I photocleavage of a ketone-substituted nucleotide yields direct strand breaks via cleavage of the β-phosphate. The reactivity of 1 in different sequences and under a variety of conditions suggests that the rate constant for strand scission is significantly greater than 106 s–1 at pH 7.2. The initially formed C2′-radical (1) is not trapped under a variety of conditions, consistent with computational studies ( Chem.—Eur. J. 2009, 15, 2394) that suggest that the barrier to strand scission is very low and that synchronous proton transfer from the 2′-hydroxyl to the departing phosphate group facilitates cleavage. The C2′-radical could be a significant contributor to RNA strand scission by the hydroxyl radical, particularly under anaerobic conditions where 1 can be produced from nucleobase radicals.
Co-reporter:Rakesh Paul
Journal of the American Chemical Society 2015 Volume 137(Issue 2) pp:596-599
Publication Date(Web):January 12, 2015
DOI:10.1021/ja511401g
C2′-Nucleotide radicals have been proposed as key intermediates in direct strand break formation in RNA exposed to ionizing radiation. Uridin-2′-yl radical (1) was independently generated in single- and double-stranded RNA via photolysis of a ketone precursor. Direct stand breaks result from heterolytic cleavage of the adjacent C3′-carbon–oxygen bond. Trapping of 1 by O2 or β-mercaptoethanol (1 M) does not compete with strand scission, indicating that phosphate elimination is >106 s–1. Uracil loss also does not compete with strand scission. When considered in conjunction with reports that nucleobase radicals produce 1, this chemistry explains why RNA is significantly more susceptible to strand scission by ionizing radiation (hydroxyl radical) than is DNA.
Co-reporter:Liwei Weng
Journal of the American Chemical Society 2015 Volume 137(Issue 34) pp:11022-11031
Publication Date(Web):August 20, 2015
DOI:10.1021/jacs.5b05478
C5′-Hydrogen atoms are frequently abstracted during DNA oxidation. The oxidized abasic lesion 5′-(2-phosphoryl-1,4-dioxobutane) (DOB) is an electrophilic product of the C5′-radical. DOB is a potent irreversible inhibitor of DNA polymerase β, and forms interstrand cross-links in free DNA. We examined the reactivity of DOB within nucleosomes and nucleosome core particles (NCPs), the monomeric component of chromatin. Depending upon the position at which DOB is generated within a NCP, it is excised from nucleosomal DNA at a rate 275–1500-fold faster than that in free DNA. The half-life of DOB (7.0–16.8 min) in NCPs is shorter than any other abasic lesion. DOB’s lifetime in NCPs is also significantly shorter than the estimated lifetime of an abasic site within a cell, suggesting that the observed chemistry would occur intracellularly. Histones also catalyze DOB excision when the lesion is present in the DNA linker region of a nucleosome. Schiff-base formation between DOB and histone proteins is detected in nucleosomes and NCPs, resulting in pyrrolone formation at the lysine residues. The lysines modified by DOB are often post-translationally modified. Consequently, the histone modifications described herein could affect the regulation of gene expression and may provide a chemical basis for the cytotoxicity of the DNA damaging agents that produce this lesion.
Co-reporter:Liwei Weng, Chuanzheng Zhou, and Marc M. Greenberg
ACS Chemical Biology 2015 Volume 10(Issue 2) pp:622
Publication Date(Web):December 5, 2014
DOI:10.1021/cb500737y
The histone proteins in nucleosome core particles are known to catalyze DNA cleavage at abasic and oxidized abasic sites, which are produced by antitumor antibiotics and as a consequence of other modalities of DNA damage. The lysine rich histone tails whose post-translational modifications regulate genetic expression in cells are mainly responsible for this chemistry. Cleavage at a C4′-oxidized abasic site (C4-AP) concomitantly results in modification of lysine residues in histone tails. Using LC-MS/MS, we demonstrate here that that Lys8, -12, -16, and -20 of histone H4 were modified when C4-AP was incorporated at a hot spot (superhelical location 1.5) for DNA damage within a nucleosome core particle. A new DNA–protein cross-linking method that provides a more quantitative analysis of individual amino acid reactivity is also described. DNA–protein cross-links were produced by an irreversible reaction between a nucleic acid electrophile that was produced following oxidatively induced rearrangement of a phenyl selenide derivative of thymidine (3) and nucleophilic residues within proteins. In addition to providing high yields of DNA–protein cross-links, kinetic analysis of the cross-linking reaction yielded rate constants that enabled ranking the contributions by individual or groups of amino acids. Cross-linking from 3 at superhelical location 1.5 revealed the following order of reactivity for the nucleophilic amino acids in the histone H4 tail: His18 > Lys16 > Lys20 ≈ Lys8, Lys12 > Lys5. Cross-linking via 3 will be generally useful for investigating DNA–protein interactions.
Co-reporter:Souradyuti Ghosh and Marc M. Greenberg
Biochemistry 2015 Volume 54(Issue 40) pp:6274-6283
Publication Date(Web):October 1, 2015
DOI:10.1021/acs.biochem.5b00860
C4′-oxidized (C4-AP) and C5′-oxidized abasic sites (DOB) that are produced following abstraction of a hydrogen atom from the DNA backbone reversibly form cross-links selectively with dA opposite a 3′-adjacent nucleotide, despite the comparable proximity of an opposing dA. A previous report on UvrABC incision of DNA substrates containing stabilized analogues of the ICLs derived from C4-AP and DOB also indicated that the latter is repaired more readily by nucleotide excision repair [Ghosh, S., and Greenberg, M. M. (2014) Biochemistry 53, 5958–5965]. The source for selective cross-link formation was probed by comparing the reactivity of ICL analogues of C4-AP and DOB that mimic the preferred and disfavored cross-links with that of reagents that indirectly detect distortion by reacting with the nucleobases. The disfavored C4-AP and DOB analogues were each more reactive than the corresponding preferred cross-link substrates, suggesting that the latter are more stable, which is consistent with selective ICL formation. In addition, the preferred DOB analogue is more reactive than the respective C4-AP ICL, which is consistent with its more efficient incision by UvrABC. The conclusions drawn from the chemical probing experiments are corroborated by UV melting studies. The preferred ICLs exhibit melting temperatures higher than those of the corresponding disfavored isomers. These studies suggest that oxidized abasic sites form reversible interstrand cross-links with dA opposite the 3′-adjacent thymidine because these products are more stable and the thermodynamic preference is reflected in the transition states for their formation.
Co-reporter:Marisa L. Taverna Porro and Marc M. Greenberg
Chemical Research in Toxicology 2015 Volume 28(Issue 4) pp:810
Publication Date(Web):March 9, 2015
DOI:10.1021/acs.chemrestox.5b00032
Double-strand breaks are widely accepted to be the most toxic form of DNA damage. Molecules that produce double-strand breaks via a single chemical event are typically very cytotoxic and far less common than those that form single-strand breaks. It was recently reported that a commonly formed C4′-radical produces double-strand breaks under aerobic conditions. Experiments described herein indicate that a peroxyl radical initiates strand damage on the complementary strand via C4′-hydrogen atom abstraction. Inferential evidence suggests that a C3′-peroxyl radical induces complementary strand damage more efficiently than does a C4′-peroxyl radical. Complementary strand hydrogen atom abstraction by the peroxyl radical is efficiently quenched by thiols. This mechanism could contribute to the higher than expected yield of double-strand breaks produced by ionizing radiation.
Co-reporter:In Seok Hong, Marc M. Greenberg
Bioorganic & Medicinal Chemistry Letters 2015 Volume 25(Issue 21) pp:4918-4921
Publication Date(Web):1 November 2015
DOI:10.1016/j.bmcl.2015.05.042
8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) is a commonly formed DNA lesion that is useful as a biomarker for oxidative stress. Methods for detecting 8-oxodGuo at specific positions within DNA could be useful for correlating DNA damage with mutational hotspots and repair enzyme accessibility. We describe a method for covalently linking (‘tagging’) peptide nucleic acids (PNAs) containing terminal nucleophiles under oxidative conditions to 8-oxodGuo at specific sites within DNA. Several nucleophiles were examined and the ε-amine of lysine was selected for further studies. As little as 10 fmol of 8-oxodGuo were detected by gel shift using 32P-labeled target DNA and no tagging of dG at the same site or 8-oxodGuo at a distal site was detected when potassium ferricyanide was used as oxidant in substrates as long as 221 bp.
Co-reporter:Arnab Rudra, Dianjie Hou, Yonggang Zhang, Jonathan Coulter, Haoming Zhou, Theodore L. DeWeese, and Marc M. Greenberg
The Journal of Organic Chemistry 2015 Volume 80(Issue 21) pp:10675-10685
Publication Date(Web):October 28, 2015
DOI:10.1021/acs.joc.5b01833
Ionizing radiation is frequently used to kill tumor cells. However, hypoxic solid tumor cells are more resistant to this treatment, providing the impetus to develop molecules that sensitize cells to ionizing radiation. 5-Bromo-2′-deoxyuridine (BrdU) has been investigated as a radiosensitizing agent in the lab and clinic for almost 5 decades. Recent reports that BrdU yields DNA interstrand cross-links (ICLs) in non-base-paired regions motivated us to develop radiosensitizing agents that generate cross-links in duplex DNA selectively under anoxic conditions. 4-Bromo- and 5-bromopyridone analogues of BrdU were synthesized and incorporated into oligonucleotides via solid-phase synthesis. Upon irradiation, these molecules yield DNA interstrand cross-links under anaerobic conditions. The respective nucleotide triphosphates are substrates for some DNA polymerases. ICLs are produced upon irradiation under anoxic conditions when the 4-bromopyridone is present in a PCR product. Because the nucleoside analogue is a poor phosphorylation substrate for human deoxycytidine kinase, a pro-nucleotide form of the 4-bromopyridone was used to incorporate this analogue into cellular DNA. Despite these efforts, the 4-bromopyridone nucleotide was not detected in cellular DNA. Although these molecules are improvements over previously reported nucleotide analogues designed to be hypoxic radiosensitizing agents, additional advances are needed to create molecules that function in cells.
Co-reporter:Dr. Subrata Panja;Dr. Rakesh Paul; Marc M. Greenberg; Sarah A. Woodson
Angewandte Chemie International Edition 2015 Volume 54( Issue 25) pp:7281-7284
Publication Date(Web):
DOI:10.1002/anie.201501658
Abstract
Non-coding antisense RNAs regulate bacterial genes in response to nutrition or environmental stress, and can be engineered for artificial gene control. The RNA chaperone Hfq accelerates antisense pairing between non-coding RNAs and their mRNA targets, by a mechanism still unknown. We used a photocaged guanosine derivative in an RNA oligonucleotide to temporally control Hfq catalyzed annealing. Using a fluorescent molecular beacon as a reporter, we observed RNA duplex formation within 15 s following irradiation (3 s) of photocaged RNA complexed with Hfq. The results showed that the Hfq chaperone directly stabilizes the initiation of RNA base pairs, and suggests a strategy for light-activated control of gene expression by non-coding RNAs.
Co-reporter:Dr. Subrata Panja;Dr. Rakesh Paul; Marc M. Greenberg; Sarah A. Woodson
Angewandte Chemie 2015 Volume 127( Issue 25) pp:7389-7392
Publication Date(Web):
DOI:10.1002/ange.201501658
Abstract
Non-coding antisense RNAs regulate bacterial genes in response to nutrition or environmental stress, and can be engineered for artificial gene control. The RNA chaperone Hfq accelerates antisense pairing between non-coding RNAs and their mRNA targets, by a mechanism still unknown. We used a photocaged guanosine derivative in an RNA oligonucleotide to temporally control Hfq catalyzed annealing. Using a fluorescent molecular beacon as a reporter, we observed RNA duplex formation within 15 s following irradiation (3 s) of photocaged RNA complexed with Hfq. The results showed that the Hfq chaperone directly stabilizes the initiation of RNA base pairs, and suggests a strategy for light-activated control of gene expression by non-coding RNAs.
Co-reporter:Marc M. Greenberg
Accounts of Chemical Research 2014 Volume 47(Issue 2) pp:646
Publication Date(Web):December 26, 2013
DOI:10.1021/ar400229d
Abasic lesions are a family of DNA modifications that lack Watson–Crick bases. The parent member of this family, the apurinic/apyrimidinic lesion (AP), occurs as an intermediate during DNA repair, following nucleobase alkylation, and from random hydrolysis of native nucleotides. In a given day, each cell produces between 10000 and 50000 AP lesions. A variety of oxidants including γ-radiolysis produce oxidized abasic sites, such as C4-AP, from the deoxyribose backbone. A number of potent, cytotoxic antitumor agents, such as bleomycin and the enediynes (e.g., calicheamicin, esperamicin, and neocarzinostatin) also lead to oxidized abasic sites in DNA.The absence of Watson–Crick bases prevents DNA polymerases from properly determining which nucleotide to incorporate opposite abasic lesions. Consequently, several studies have revealed that (oxidized) abasic sites are highly mutagenic. Abasic lesions are also chemically unstable, are prone to strand scission, and possess electrophilic carbonyl groups. However, researchers have only uncovered the consequences of the inherent reactivity of these electrophiles within the past decade. The development of solid phase synthesis methods for oligonucleotides that both place abasic sites in defined positions and circumvent their inherent alkaline lability has facilitated this research.Chemically synthesized oligonucleotides containing abasic lesions provide substrates that have allowed researchers to discover a range of interesting chemical properties of potential biological importance. For instance, abasic lesions form DNA–DNA interstrand cross-links, a particularly important family of DNA damage because they block replication and transcription absolutely. In addition, bacterial repair enzymes can convert an interstrand cross-link derived from C4-AP into a double-strand break, the most deleterious form of DNA damage. Oxidized abasic lesions can also inhibit DNA repair enzymes that remove damaged nucleotides. DNA polymerase β, an enzyme that is irreversibly inactivated, is vitally important in base excision repair and is overproduced in some tumor cells. Nucleosome core particles, the monomeric components that make up chromatin, accentuate the chemical instability of abasic lesions. In experiments using synthetic nucleosome core particles containing abasic sites, the histone proteins catalyze strand cleavage at the sites that incorporate these lesions. Furthermore, in the presence of the C4-AP lesion, strand scission is accompanied by modification of the histone protein.The reactivity of (oxidized) abasic lesions illustrates how seemingly simple nucleic acid modifications can have significant biochemical effects and may provide a chemical basis for the cytotoxicity of the chemotherapeutic agents that produce them.
Co-reporter:Chuanzheng Zhou
Journal of the American Chemical Society 2014 Volume 136(Issue 18) pp:6562-6565
Publication Date(Web):April 22, 2014
DOI:10.1021/ja501285s
Although DNA binding proteins shield the genetic material from diffusible reactive oxygen species by reacting with them, the resulting protein (peroxyl) radicals can oxidize the bound DNA. To explore this possible DNA damage by protein radicals, histone H4 proteins containing an azoalkane radical precursor at defined sites were prepared. Photolysis of a nucleosome core particle containing the modified protein produces DNA damage that is consistent with selective C4′-oxidation. The nucleotide(s) damaged is highly dependent on proximity to the protein radical. These experiments provide insight into the effects of oxidative stress on protein-bound DNA, revealing an additional layer of complexity concerning nucleic acid damage.
Co-reporter:Dumitru Arian ; Mohammad Hedayati ; Haoming Zhou ; Zoe Bilis ; Karen Chen ; Theodore L. DeWeese
Journal of the American Chemical Society 2014 Volume 136(Issue 8) pp:3176-3183
Publication Date(Web):February 11, 2014
DOI:10.1021/ja411733s
Abasic sites are ubiquitous DNA lesions that are mutagenic and cytotoxic but are removed by the base excision repair pathway. DNA polymerase β carries out two of the four steps during base excision repair, including a lyase reaction that removes the abasic site from DNA following incision of its 5′-phosphate. DNA polymerase β is overexpressed in cancer cells and is a potential anticancer target. Recently, DNA oxidized abasic sites that are produced by potent antitumor agents were shown to inactivate DNA polymerase β. A library of small molecules whose structures were inspired by the oxidized abasic sites was synthesized and screened for the ability to irreversibly inhibit DNA polymerase β. One candidate (3a) was examined more thoroughly, and modification of its phosphate backbone led to a molecule that irreversibly inactivates DNA polymerase β in solution (IC50 ≈ 21 μM), and inhibits the enzyme’s lyase activity in cell lysates. A bisacetate analogue is converted in cell lysates to 3a. The bisacetate is more effective in cell lysates, more cytotoxic in prostate cancer cells than 3a and potentiates the cytotoxicity of methyl methanesulfonate between 2- and 5-fold. This is the first example of an irreversible inhibitor of the lyase activity of DNA polymerase β that works synergistically with a DNA damaging agent.
Co-reporter:Joanna Maria N. San Pedro
Journal of the American Chemical Society 2014 Volume 136(Issue 10) pp:3928-3936
Publication Date(Web):February 28, 2014
DOI:10.1021/ja412562p
Nucleobase radicals are a major family of reactive species produced in DNA as a result of oxidative stress. Two such radicals, 5-hydroxy-5,6-dihydrothymidin-6-yl radical (1) and 5,6-dihydrouridin-6-yl radical (5), were independently generated within chemically synthesized oligonucleotides from photochemical precursors. Neither nucleobase radical produces direct strand breaks or alkali-labile lesions in single or double stranded DNA. The respective peroxyl radicals, resulting from O2 trapping, add to 5′-adjacent nucleobases, with a preference for dG. Distal dG’s are also oxidatively damaged by the peroxyl radicals. Experiments using a variety of sequences indicate that distal damage occurs via covalent modification of the 5′-adjacent dG, but there is no evidence for electron transfer by the nucleobase peroxyl radicals.
Co-reporter:Souradyuti Ghosh and Marc M. Greenberg
Biochemistry 2014 Volume 53(Issue 37) pp:
Publication Date(Web):September 10, 2014
DOI:10.1021/bi500914d
Nucleotide excision repair is a primary pathway in cells for coping with DNA interstrand cross-links (ICLs). Recently, C4′-oxidized (C4-AP) and C5′-oxidized abasic sites (DOB) that are produced following hydrogen atom abstraction from the DNA backbone were found to produce ICLs. Because some of the ICLs derived from C4-AP and DOB are too unstable to characterize in biochemical processes, chemically stable analogues were synthesized [Ghosh, S., and Greenberg, M. M. (2014) J. Org. Chem. 79, 5948–5957]. UvrABC incision of DNA substrates containing stabilized analogues of the ICLs derived from C4-AP and DOB was examined. The incision pattern for the ICL related to the C4′-oxidized abasic site was typical for UvrABC substrates. UvrABC cleaved both strands of the substrate containing the C4-AP ICL analogue, but it was a poor substrate. UvrABC incised <30% of the C4-AP ICL analogue over an 8 h period, raising the possibility that this cross-link will be inefficiently repaired in cells. Furthermore, double-strand breaks were not detected upon incision of an internally labeled hairpin substrate containing the C4-AP ICL analogue. UvrABC incised the stabilized analogue of the DOB ICL more efficiently (∼20% in 1 h). Furthermore, the incision pattern was unique, and the cross-linked substrate was converted into a single product, a double-strand break. The template strand was exclusively incised on the template strand on the 3′-side of the cross-linked dA. Although the outcomes of the interaction between UvrABC and these two cross-linked substrates are different from one another, they provide additional examples of how seemingly simple lesions (C4-AP and DOB) can potentially exert significant deleterious effects on biochemical processes.
Co-reporter:John Ernest Vallarta Bajacan, In Seok Hong, Trevor W. Penning, and Marc M. Greenberg
Chemical Research in Toxicology 2014 Volume 27(Issue 7) pp:1227
Publication Date(Web):June 16, 2014
DOI:10.1021/tx500120p
8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) is a commonly formed DNA lesion that is useful as a biomarker for oxidative stress. Although methods for selective quantification of 8-oxodGuo exist, there is room for additional methods that are sensitive and utilize instrumentation that is widely available. We previously took advantage of the reported reactivity of 8-oxodGuo to develop a method for detecting the lesion by selectively covalently tagging it with a molecule equipped with a biotin label that can be used subsequently with a reporting method (Xue, L. and Greenberg, M. M. (2007) J. Am. Chem. Soc.129, 7010). We now report a method that can detect as little as 14 amol of 8-oxodGuo by tagging DNA with a reagent containing a disulfide that reduces background due to nonspecific binding. The reagent also contains biotin that enables capturing target DNA on streptavidin-coated magnetic beads. The captured DNA is quantified using quantitative PCR. The method is validated by comparing the amount of 8-oxodGuo detected as a function of Fe2+/H2O2/ascorbate-dose to that reported previously using mass spectrometry.
Co-reporter:Dianjie Hou and Marc M. Greenberg
The Journal of Organic Chemistry 2014 Volume 79(Issue 5) pp:1877-1884
Publication Date(Web):February 24, 2014
DOI:10.1021/jo4028227
γ-Radiolysis kills cells by damaging DNA via radical processes. Many of the radical pathways are O2 dependent, which results in a reduction in the cytotoxicity of ionizing radiation in hypoxic tumor cells. Consequently, there is a need for chemical agents that increase DNA damage by ionizing radiation under O2-deficient conditions. Modified nucleotides that are incorporated in DNA and produce highly reactive σ-radicals are useful as radiosensitizing agents. Aryl halide C-nucleotides (4–6) were incorporated into oligonucleotides by solid-phase synthesis. Duplex DNA containing 4–6 forms interstrand cross-links upon γ-radiolysis under anaerobic conditions or UV irradiation. Deep Vent (exo–) DNA polymerase accepted the nucleotide triphosphate of C-nucleotide 6 as a substrate and preferentially incorporated it opposite pyrimidines, but no further extension was detected. Incorporation of 6 in extended products by Deep Vent (exo–) during PCR or by Sequenase during copying of single stranded DNA plasmid was undetectable. Aryl halide nucleotide analogues that produce DNA interstrand cross-links under anaerobic conditions upon irradiation are potentially useful as radiosensitizing agents, but further research is needed to identify molecules that are incorporated by DNA polymerases and do not block further polymerization for this approach to be useful in cells.
Co-reporter:Souradyuti Ghosh and Marc M. Greenberg
The Journal of Organic Chemistry 2014 Volume 79(Issue 13) pp:5948-5957
Publication Date(Web):June 20, 2014
DOI:10.1021/jo500944g
DNA interstrand cross-links are an important family of DNA damage that block replication and transcription. Recently, it was discovered that oxidized abasic sites react with the opposing strand of DNA to produce interstrand cross-links. Some of the cross-links between 2′-deoxyadenosine and the oxidized abasic sites, 5′-(2-phosphoryl-1,4-dioxobutane) (DOB) and the C4-hydroxylated abasic site (C4-AP), are formed reversibly. Chemical instability hinders biochemical, structural, and physicochemical characterization of these cross-linked duplexes. To overcome these limitations, we developed methods for preparing stabilized analogues of DOB and C4-AP cross-links via solid-phase oligonucleotide synthesis. Oligonucleotides of any sequence are attainable by synthesizing phosphoramidites in which the hydroxyl groups of the cross-linked product were orthogonally protected using photochemically labile and hydrazine labile groups. Selective unmasking of a single hydroxyl group precedes solid-phase synthesis of one arm of the cross-linked DNA. The method is compatible with commercially available phosphoramidites and other oligonucleotide synthesis reagents. Cross-linked duplexes containing as many as 54 nt were synthesized on solid-phase supports. Subsequent enzyme ligation of one cross-link product provided a 60 bp duplex, which is suitable for nucleotide excision repair studies.
Co-reporter:Jack L. Sloane and Marc M. Greenberg
The Journal of Organic Chemistry 2014 Volume 79(Issue 20) pp:9792-9798
Publication Date(Web):October 8, 2014
DOI:10.1021/jo501982r
RNA oligonucleotides containing a phenyl selenide derivative of 5-methyluridine were chemically synthesized by solid-phase synthesis. The phenyl selenide is rapidly converted to an electrophilic, allylic phenyl seleneate under mild oxidative conditions. The phenyl seleneate yields interstrand cross-links when part of a duplex and is useful for synthesizing oligonucleotide conjugates. Formation of the latter is illustrated by reaction of an oligonucleotide containing the phenyl selenide with amino acids in the presence of mild oxidant. The products formed are analogous to those observed in tRNA that are believed to be formed posttranslationally via a biosynthetic intermediate that is chemically homologous to the phenyl seleneate.
Co-reporter:Rakesh Paul and Marc M. Greenberg
The Journal of Organic Chemistry 2014 Volume 79(Issue 21) pp:10303-10310
Publication Date(Web):October 17, 2014
DOI:10.1021/jo501916r
The uridin-2′-yl radical (1) has been proposed as an intermediate during RNA oxidation. However, its reactivity has not been thoroughly studied due to the complex conditions under which it is typically generated. The uridin-2′-yl radical was independently generated from a benzyl ketone (2a) via Norrish type I photocleavage upon irradiation at λmax = 350 nm. Dioxygen and β-mercaptoethanol are unable to compete with loss of uracil from 1 in phosphate buffer. Thiol trapping competes with uracil fragmentation in less polar solvent conditions. This is ascribed mostly to a reduction in the rate constant for uracil elimination in the less polar solvent. Hydrogen atom transfer to 1 from β-mercaptoethanol occurs exclusively from the α-face to produce arabinouridine. Mass balances range from 72 to 95%. Furthermore, the synthesis of 2a is amenable to formation of the requisite phosphoramidite for solid-phase oligonucleotide synthesis. This and the fidelity with which the urdin-2′-yl radical is generated from 2a suggest that this precursor should be useful for studying the radical’s reactivity in synthetic oligonucleotides.
Co-reporter:Chuanzheng Zhou ; Jonathan T. Sczepanski
Journal of the American Chemical Society 2013 Volume 135(Issue 14) pp:5274-5277
Publication Date(Web):March 26, 2013
DOI:10.1021/ja400915w
The C4′-oxidized abasic site is produced in DNA by a variety of oxidizing agents, including potent cytotoxic antitumor agents. Independent generation of this alkali-labile lesion at defined positions within nucleosome core particles reveals that the histone proteins increase strand scission between 130- and 550-fold. Strand scission proceeds via a Schiff base intermediate, but the DNA–protein cross-links are unstable. The oxidized abasic site is removed in its entirety from the DNA and transferred to the lysine-rich tail region of the proximal histone protein in the form of a lactam. The modification is distributed over several residues within the amino-terminal tail of the proximal histone. Transfer of DNA damage to histones could affect gene regulation.
Co-reporter:Marisa L. Taverna Porro
Journal of the American Chemical Society 2013 Volume 135(Issue 44) pp:16368-16371
Publication Date(Web):October 22, 2013
DOI:10.1021/ja409513q
Double strand breaks (DSBs) are the most deleterious form of DNA damage. Natural products that produce them are potent cytotoxic agents. Designing molecules that produce DSBs via a single chemical event is challenging. We determined that formation of a C4′-nucleotide radical in duplex DNA under aerobic conditions gives rise to a DSB. The original radical yields a strand break containing a peroxyl radical, which initiates opposite strand cleavage via C4′-hydrogen atom abstraction. This mechanism provides the impetus to design DNA damaging agents that produce DSBs by abstracting a single hydrogen atom from the biopolymer.
Co-reporter:Liwei Weng, Sonia M. Horvat, Carl H. Schiesser, and Marc M. Greenberg
Organic Letters 2013 Volume 15(Issue 14) pp:3618-3621
Publication Date(Web):July 3, 2013
DOI:10.1021/ol401472m
Generation of the 5-(2′-deoxyuridinyl)methyl radical (6) was reexamined. Trapping by 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl confirms that 6 is generated. However, trapping by methoxyamine reveals that the respective carbocation (10) is also produced. Examining the effects of these traps on products in DNA reveals that the carbocation and not 6 yields interstrand cross-links. Cross-link formation from the carbocation is consistent with DFT calculations that predict that addition at the N1 position of dA is essentially barrierless.
Co-reporter:John Ernest V. Bajacan and Marc M. Greenberg
Biochemistry 2013 Volume 52(Issue 37) pp:
Publication Date(Web):September 9, 2013
DOI:10.1021/bi400997h
Translesion synthesis past an oxidized abasic site, 2-deoxyribonolactone, in Escherichia coli results in high levels of dG incorporation and is dependent upon DNA polymerase V (Pol V). Kinetic experiments performed here affirm that Pol V preferentially incorporates dG opposite 2-deoxyribonolactone (L). Pol V discriminates between dG and dA on the basis of the apparent KD, suggesting that L provides instructive structural information to the enzyme despite lacking a Watson–Crick base.
Co-reporter:Adam J. Stevens, Lirui Guan, Katarzyna Bebenek, Thomas A. Kunkel, and Marc M. Greenberg
Biochemistry 2013 Volume 52(Issue 5) pp:
Publication Date(Web):January 18, 2013
DOI:10.1021/bi301592x
Base excision repair (BER) plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase λ (Pol λ) is believed to play a backup role to DNA polymerase β (Pol β) in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA-damaging agents, including several antitumor antibiotics, the C4′-oxidized abasic site following Ape1 incision (pC4-AP), and 5′-(2-phosphoryl-1,4-dioxobutane) (DOB), irreversibly inactivate Pol β and Pol λ. The interactions of DOB and pC4-AP with Pol λ are examined in detail using DNA substrates containing these lesions at defined sites. Single-turnover kinetic experiments show that Pol λ excises DOB almost 13 times more slowly than a 5′-phosphorylated 2-deoxyribose (dRP). pC4-AP is excised approximately twice as fast as DOB. The absolute rate constants are considerably slower than those reported for Pol β for the respective reactions, suggesting that Pol λ may be an inefficient backup in BER. DOB inactivates Pol λ approximately 3-fold less efficiently than it does Pol β, and the difference can be attributed to a higher KI (33 ± 7 nM). Inactivation of Pol λ’s lyase activity by DOB also prevents the enzyme from conducting polymerization following preincubation of the protein and DNA. Mass spectral analysis of GluC-digested Pol λ inactivated by DOB shows that Lys324 is modified. There is inferential support for the idea that Lys312 may also be modified. Both residues are within the Pol λ lyase active site. When acting on pC4-AP, Pol λ achieves approximately four turnovers on average before being inactivated. Lyase inactivation by pC4-AP is also accompanied by loss of polymerase activity, and mass spectrometry indicates that Lys312 and Lys324 are modified by the lesion. The ability of DOB and pC4-AP to inactivate Pol λ provides additional evidence that these lesions are significant sources of the cytotoxicity of DNA-damaging agents that produce them.
Co-reporter:Jonathan T. Sczepanski, Chuanzheng Zhou, and Marc M. Greenberg
Biochemistry 2013 Volume 52(Issue 12) pp:
Publication Date(Web):March 12, 2013
DOI:10.1021/bi3010076
The reactivity of apurinic/apyrimidinic (AP) sites at different locations within nucleosome core particles was examined. AP sites are greatly destabilized in nucleosome core particles compared to free DNA. Their reactivity varied ∼5-fold with respect to the location within the nucleosome core particles but followed a common mechanism involving formation of a Schiff base between histone proteins and the lesion. The identity of the histone protein(s) involved in the reaction and the reactivity of the corresponding DNA–protein cross-links varied with the location of the abasic site, indicating that while the relative rate constants for individual steps varied in a complex manner, the overall mechanism remained the same. The source of the accelerated reactivity was probed using nucleosomes containing AP89 and histone H3 and H4 variants. Mutating the five lysine residues in the amino tail region of histone H4 to arginines reduced the rate constant for disappearance almost 15-fold. Replacing histidine 18 with an alanine reduced AP reactivity more than 3-fold. AP89 in a nucleosome core particle composed of the H4 variant containing both sets of mutations reacted only <4-fold faster than it did in naked DNA. These experiments reveal that nucleosome-catalyzed reaction at AP89 is a general phenomenon and that the lysine rich histone tails, whose modification is integrally involved in epigenetics, are primarily responsible for this chemistry.
Co-reporter:Joanna Maria N. San Pedro ; Dr. Marc M. Greenberg
ChemBioChem 2013 Volume 14( Issue 13) pp:1590-1596
Publication Date(Web):
DOI:10.1002/cbic.201300369
Abstract
Photolysis of an aryl sulfide-containing 5,6-dihydropyrimidine (1) at 350 nm produces high yields of thymidine and products resulting from trapping of a 5,6-dihydrothymidin-5-yl radical by O2 or thiols. Thymidine is believed to result from disproportionation of the radical pair originally generated from CS bond homolysis of 1 on the microsecond timescale, which is significantly shorter than other photochemical transformations of modified nucleotides into their native forms. Duplex DNA containing 1 is destabilized, presumably due to disruption of π-stacking. Incorporation of 1 within the binding site of the restriction endonuclease EcoRV provides a photochemical switch for turning on the enzyme's activity. In contrast, 1 is a substrate for endonuclease VIII and serves as a photochemical off switch for this base excision repair enzyme. Modification 1 also modulates the activity of the 10–23 DNAzyme, despite its incorporation into a nonduplex region. Overall, dihydropyrimidine 1 shows promise as a tool to provide spatiotemporal control over DNA structure on the miscrosecond timescale.
Co-reporter:Marc M. Greenberg
Accounts of Chemical Research 2012 Volume 45(Issue 4) pp:588
Publication Date(Web):November 11, 2011
DOI:10.1021/ar2002182
DNA is constantly exposed to agents that induce structural damage, from sources both internal and external to an organism. Endogenous species, such as oxidizing chemicals, and exogenous agents, such as ultraviolet rays in sunlight, together produce more than 70 distinct chemical modifications of native nucleotides. Of these, about 15 of the lesions have been detected in cellular DNA. This kind of structural DNA damage can be cytotoxic, carcinogenic, or both and is being linked to an increasingly lengthy list of diseases.The formamidopyrimidine (Fapy) lesions are a family of DNA lesions that result after purines undergo oxidative stress. The Fapy lesions are produced in yields comparable to the 8-oxopurines, which, owing in part to a perception of mutagenicity in some quarters, have been subjected to intense research scrutiny. But despite the comparable abundance of the formamidopyrimidines and the 8-oxopurines, until recently very little was known about the effects of Fapy lesions on biochemical processes involving DNA or on the structure and stability of the genomic material.In this Account, we discuss the detection of Fapy lesions in DNA and the mechanism proposed for their formation. We also describe methods for the chemical synthesis of oligonucleotides containing Fapy·dA or Fapy·dG and the outcomes of chemical and biochemical studies utilizing these compounds. These experiments reveal that the formamidopyrimidines decrease the fidelity of polymerases and are substrates for DNA repair enzymes. The mutation frequency of Fapy·dG in mammals is even greater than that of 8-oxodGuo (8-oxo-7,8-dihydro-2′-deoxyguanosine, one of the 8-oxopurines), suggesting that this lesion could be a useful biomarker and biologically significant.Despite clear similarities, the formamidopyrimidines have lived in the shadow of the corresponding 8-oxopurine lesions. But the recent development of methods for synthesizing oligonucleotides containing Fapy·dA or Fapy·dG has accelerated research on these lesions, revealing that the formamidopyrimidines are repaired as efficiently and, in some cases, more rapidly than the 8-oxopurines. Fapy·dG appears to be a lesion of biochemical consequence, and further study of its mutagenicity, repair, and interactions with DNA structure will better define the cellular details involving this important product of DNA stress.
Co-reporter:Chuanzheng Zhou ; Jonathan T. Sczepanski
Journal of the American Chemical Society 2012 Volume 134(Issue 40) pp:16734-16741
Publication Date(Web):September 28, 2012
DOI:10.1021/ja306858m
Duplex DNA containing an apurinic/apyrimidinic (AP) lesion undergoes cleavage significantly more rapidly in nucleosome core particles (NCPs) than it does when free. The mechanism of AP cleavage within NCPs was studied through independently generating lesions within them. AP mediated DNA cleavage within NCPs is initiated by DNA–protein cross-link (DPCun) formation followed by β-elimination to give DPCs containing cleaved DNA (DPCcl). Hydrolysis of DPCcl produces a DNA single strand break (SSB). C2-dideuteration of AP showed that deprotonation from this position is involved in the rate-determining step. Experiments utilizing NCPs containing mutated histone H4 proteins indicated that lysine residues in the amino terminal tail are involved in both DPC formation and β-elimination steps. Lysines 16 and 20 seem to play a greater role in reacting with AP at superhelical location 1.5, but other amino acids (e.g., lysines 5, 8, and 12) compensate in their absence. The mechanism of rapid double strand breaks in bistranded, clustered AP lesions was studied by independently preparing reaction intermediates within model NCPs. A single strand break on one strand enhances the cleavage of a proximal AP on the opposite strand.
Co-reporter:Marino J. E. Resendiz ; Arne Schön ; Ernesto Freire
Journal of the American Chemical Society 2012 Volume 134(Issue 30) pp:12478-12481
Publication Date(Web):July 24, 2012
DOI:10.1021/ja306304w
Photolabile nucleotides that disrupt nucleic acid structure are useful mechanistic probes and can be used as tools for regulating biochemical processes. Previous probes can be limited by the need to incorporate multiple modified nucleotides into oligonucleotides and in kinetic studies by the rate-limiting step in the conversion to the native nucleotide. Photolysis of aryl sulfide 1 produces high yields of 5-methyluridine, and product formation is complete in less than a microsecond. Aryl sulfide 1 prevents RNA hairpin formation and complete folding of the preQ1 class I riboswitch. Proper folding is achieved in each instance upon photolysis at 350 nm. Aryl sulfide 1 is a novel tool for modulating RNA structure, and formation of 5-methyluridine within a radical cage suggests that it will be useful in kinetic studies.
Co-reporter:Marino J. E. Resendiz ; Venkata Pottiboyina ; Michael D. Sevilla
Journal of the American Chemical Society 2012 Volume 134(Issue 8) pp:3917-3924
Publication Date(Web):February 15, 2012
DOI:10.1021/ja300044e
Nucleobase radicals are the major family of reactive intermediates produced when nucleic acids are exposed to γ-radiolysis. The 5,6-dihydrouridin-5-yl radical (1), the formal product of hydrogen atom addition and a model for hydroxyl radical addition, was independently generated from a ketone precursor via Norrish Type I photocleavage in single and double stranded RNA. Radical 1 produces direct strand breaks at the 5′-adjacent nucleotide and only minor amounts of strand scission are observed at the initial site of radical generation. Strand scission occurs preferentially in double stranded RNA and in the absence of O2. The dependence of strand scission efficiency from the 5,6-dihydrouridin-5-yl radical (1) on secondary structure under anaerobic conditions suggests that this reactivity may be useful for extracting additional RNA structural information from hydroxyl radical reactions. Varying the identity of the 5′-adjacent nucleotide has little effect on strand scission. Internucleotidyl strand scission occurs via β-elimination of the 3′-phosphate following C2′-hydrogen atom abstraction by 1. The subsequently formed olefin cation radical yields RNA fragments containing 3′-phosphate or 3′-deoxy-2′-ketonucleotide termini from competing deprotonation pathways. The ketonucleotide end group is favored in the presence of low concentrations of thiol, presumably by reducing the cation radical to the enol. Competition studies with thiol show that strand scission from the 5,6-dihydrouridin-5-yl radical (1) is significantly faster than from the 5,6-dihydrouridin-6-yl radical (2) and is consistent with computational studies using the G3B3 approach that predict the latter to be more stable than 1 by 2.8 kcal/mol.
Co-reporter:Chuanzheng Zhou
Journal of the American Chemical Society 2012 Volume 134(Issue 19) pp:8090-8093
Publication Date(Web):May 2, 2012
DOI:10.1021/ja302993h
Oxidized abasic sites such as 2-deoxyribonolactone (L) are produced in DNA by a variety of oxidizing agents, including potent cytotoxic antitumor natural products. 2-Deoxyribonolactone is labile under alkaline conditions, but its half-life in free DNA at pH 7.5 is approximately 1 week. Independent generation of L at defined positions within nucleosomes reveals that the histone proteins catalyze strand scission and increase the rate between 11- and ∼43-fold. Mechanistic studies indicate that DNA–protein cross-links are not intermediates en route to strand scission and that C2 deprotonation is the rate-determining step. The use of mutant histone H4 proteins demonstrates that the lysine-rich tail that is often post-translationally modified in cells contributes to the cleavage of L but is not the sole source of the enhanced cleavage rates. Consideration of DNA repair in cells suggests that L formation in nucleosomal DNA as part of bistranded lesions by antitumor antibiotics results in de facto double strand breaks, the most deleterious form of DNA damage.
Co-reporter:Joanna Maria N. San Pedro and Marc M. Greenberg
Organic Letters 2012 Volume 14(Issue 11) pp:2866-2869
Publication Date(Web):May 22, 2012
DOI:10.1021/ol301109z
5,6-Dihydro-5-hydroxythymidin-6-yl radical (1), the major reactive intermediate resulting from hydroxyl radical addition to C5 of the pyrimidine, is produced via 350 nm photolysis of a 2,5-dimethoxyphenylsulfide precursor (2). Competition between O2 and thiol for 1 suggests that the radical reacts relatively slowly with β-mercaptoethanol compared to other alkyl radicals. Overall, aryl sulfide 2 should be an effective precursor for the major hydroxyl radical adduct of thymidine in DNA.
Co-reporter:Bintian Zhang, Liang-Hong Guo, and Marc M. Greenberg
Analytical Chemistry 2012 Volume 84(Issue 14) pp:6048
Publication Date(Web):June 18, 2012
DOI:10.1021/ac300866u
Exposure of DNA to oxidative stress conditions results in the generation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). 8-OxodGuo is genotoxic if left unrepaired. We quantified 8-oxodGuo lesions in double-stranded DNA films by using a photoelectrochemical DNA sensor in conjunction with a specific covalent labeling method. A lesion-containing DNA film was assembled on a SnO2 nanoparticle modified indium tin oxide electrode through layer-by-layer electrostatic adsorption. The lesions were covalently labeled with a biotin conjugated spermine derivative, and ruthenium tris(bipyridine) labeled streptavidin was introduced as the signal reporter molecule. Photocurrent increased with the number of lesions in the strand and decreased as the film was diluted with intact DNA. Quantification of 8-oxodGuo was achieved with an estimated detection limit of ∼1 lesion in 650 bases or 1.6 fmol of 8-oxodGuo on the electrode. Incubation of the film with a DNA base excision repair enzyme, E. coli formamidopyrimidine–DNA glycosylase (Fpg), resulted in complete loss of the signal, indicating efficient excision of the isolated lesions in the nucleotide. Oxidatively generated DNA damage to a double-stranded calf thymus DNA film by the Fenton reaction was then assessed. One 8-oxodGuo lesion in 520 bases was detected in DNA exposed to 50 μM Fe2+/200 μM H2O2. Treatment with Fpg reduced the photocurrent by 50%, indicating only partial excision of 8-oxodGuo. This suggests that tandem lesions, which are resistant to Fpg excision, are generated by the Fenton reaction. Unlike repair enzyme dependent methods, the sensor recognizes 8-oxodGuo in tandem lesions and can avoid underestimating DNA damage.
Co-reporter:Joanna Maria N. San Pedro, Terry A. Beerman, Marc M. Greenberg
Bioorganic & Medicinal Chemistry 2012 Volume 20(Issue 15) pp:4744-4750
Publication Date(Web):1 August 2012
DOI:10.1016/j.bmc.2012.06.004
C1027 is a potent antitumor agent that damages DNA. It has the unusual ability to produce double strand breaks and interstrand cross-links (ICLs) intracellularly, which enable it to initiate concurrent ataxia-telangiestasia mutated (ATM) and Rad-3 related (ATR) independent damage responses. The latter form of damage is not well characterized. We have examined the effect of DNA sequence on C1027 reactivity and found it to be more diverse than previously thought. In addition, analysis of the chemical stability of ICLs suggests that they result from reaction with the deoxyribose ring on one strand but direct addition to a nucleobase on the opposite strand.
Co-reporter:Aaron C. Jacobs ; Marino J. E. Resendiz
Journal of the American Chemical Society 2011 Volume 133(Issue 13) pp:5152-5159
Publication Date(Web):March 10, 2011
DOI:10.1021/ja200317w
Nucleobase radicals are the major reactive intermediates produced when hydroxyl radical reacts with nucleic acids. 5,6-Dihydrouridin-6-yl radical (1) was independently generated from a ketone precursor via Norrish Type I photocleavage in a dinucleotide, single-stranded, and double-stranded RNA. This radical is a model of the major hydroxyl radical adduct of uridine. Tandem lesions resulting from addition of the peroxyl radical derived from 1 to the 5′-adjacent nucleotide are observed by ESI−MS. Radical 1 produces direct strand breaks at the 5′-adjacent nucleotide and at the initial site of generation. The preference for cleavage at these two positions depends upon the secondary structure of the RNA and whether O2 is present or not. Varying the identity of the 5′-adjacent nucleotide has little effect on strand scission. In general, strand scission is significantly more efficient under anaerobic conditions than when O2 is present. Strand scission is more than twice as efficient in double-stranded RNA than in a single-stranded oligonucleotide under anaerobic conditions. Internucleotidyl strand scission occurs via β-fragmentation following C2′-hydrogen atom abstraction by 1. The subsequently formed olefin cation radical ultimately yields products containing 3′-phosphate or 3′-deoxy-2′-ketouridine termini. These end groups are proposed to result from competing deprotonation pathways. The dependence of strand scission efficiency from 1 on secondary structure under anaerobic conditions suggests that this reactivity may be useful for extracting additional RNA structural information from hydroxyl radical reactions.
Co-reporter:Aaron C. Jacobs, Cortney R. Kreller, and Marc M. Greenberg
Biochemistry 2011 Volume 50(Issue 1) pp:
Publication Date(Web):December 14, 2010
DOI:10.1021/bi1017667
The C4′-oxidized abasic site (C4-AP), which is produced by a variety of damaging agents, has significant consequences for DNA. The lesion is highly mutagenic and reactive, resulting in interstrand cross-links. The base excision repair of DNA containing independently generated C4-AP was examined. C4-AP is incised by Ape1 ∼12-fold less efficiently than an apurinic/apyrimidinic lesion. DNA polymerase β induces the β-elimination of incised C4-AP in ternary complexes, duplexes, and single-stranded substrate. However, excision from a ternary complex is most rapid. In addition, the lesion inactivates the enzyme after approximately seven turnovers on average by reacting with one or more lysine residues in the lyase active site. Unlike 5′-(2-phosphoryl-1,4-dioxobutane), which very efficiently irreversibly inhibits DNA polymerase β, the lesion is readily removed by strand displacement synthesis conducted by the polymerase in conjunction with flap endonuclease 1. DNA repair inhibition by C4-AP may be a partial cause of the cytotoxicity of drugs that produce this lesion.
Co-reporter:Jonathan T. Sczepanski, Christine N. Hiemstra, Marc M. Greenberg
Bioorganic & Medicinal Chemistry 2011 Volume 19(Issue 19) pp:5788-5793
Publication Date(Web):1 October 2011
DOI:10.1016/j.bmc.2011.08.024
The C4′-oxidized abasic site (C4-AP) forms two types of interstrand cross-links with the adjacent nucleotides in DNA. Previous experiments revealed that dG does not react with the lesion and that formation of one type of cross-link is catalyzed by the opposing dA. iso-Guanosine·dC and 2-aminopurine·dT base pairs were used to determine why dG does not cross-link with C4-AP despite its well known reactivity with other bis-electrophiles. 7-Deaza-2′-deoxyadenosine was used to probe the role of the nucleotide opposite C4-AP in the catalysis of interstrand cross-link formation.
Co-reporter:Kwan-Young Jung, Tetsuya Kodama, and Marc M. Greenberg
Biochemistry 2011 Volume 50(Issue 28) pp:
Publication Date(Web):June 22, 2011
DOI:10.1021/bi200787e
Oxidation of the C5′-position of DNA results in direct strand scission. The 3′-fragments produced contain DNA lesions at their 5′-termini. The major DNA lesion contains an aldehyde at its C5′-position, but its nucleobase is unmodified. Excision of the lesion formed from oxidation of thymidine (T-al) is achieved by strand displacement synthesis by DNA polymerase β (Pol β) in the presence or absence of flap endonuclease 1 (FEN1). Pol β displaces T-al and thymidine with comparable efficiency, but less so than a chemically stabilized abasic site analogue (F). FEN1 cleaves the flaps produced during strand displacement synthesis that are two nucleotides or longer. A ternary complex containing T-al is also a substrate for the bacterial UvrABC nucleotide excision repair system. The sites of strand scission are identical in ternary complexes containing T-al, thymidine, or F. UvrABC incision efficiency of these ternary complexes is comparable as well but significantly slower than a duplex substrate containing a bulky substituted thymidine. However, cleavage occurs only on the 5′-fragment and does not remove the lesion. These data suggest that unlike many lesions the redundant nature of base excision and nucleotide excision repair systems does not provide a means for removing the major damage product produced by agents that oxidize the C5′-position. This may contribute to the high cytotoxicity of drugs that oxidize the C5′-position in DNA.
Co-reporter:Dr. Lirui Guan;Dr. Godfried W. van der Heijden;Dr. Alex Bortvin; Dr. Marc M. Greenberg
ChemBioChem 2011 Volume 12( Issue 14) pp:2184-2190
Publication Date(Web):
DOI:10.1002/cbic.201100353
Abstract
5-Ethynyl-2′-deoxycytidine triphosphate (EdCTP) was synthesized as a probe to be used in conjunction with fluorescent labeling to facilitate the analysis of the in vivo dynamics of DNA-centered processes (DNA replication, repair and cytosine demethylation). Kinetic analysis showed that EdCTP is accepted as a substrate by Klenow exo− and DNA polymerase β. Incorporation of 5-ethynyl-2′-deoxycytidine (EdC) into DNA by these enzymes is, at most, modestly less efficient than native dC. EdC-containing DNA was visualized by using a click reaction with a fluorescent azide, following polymerase incorporation and T4 DNA ligase mediated ligation. Subsequent experiments in mouse male germ cells and zygotes demonstrated that EdC is a specific and reliable reporter of DNA replication, in vivo.
Co-reporter:Lirui Guan
Journal of the American Chemical Society 2010 Volume 132(Issue 14) pp:5004-5005
Publication Date(Web):March 24, 2010
DOI:10.1021/ja101372c
DNA damage is a source of carcinogenicity and is also the source of the cytotoxicity of γ-radiolysis and antitumor agents, such as the enediynes. The dioxobutane lesion (DOB) is produced by a variety of DNA-damaging agents, including the aforementioned. Repair of DOB is important for maintaining the integrity of the genome as well as counteracting therapeutic agents that target DNA. We demonstrate that the DOB lesion efficiently and irreversibly inhibits repair by DNA polymerase β (Pol β), an integral enzyme in base-excision repair. Irreversible inhibition of Pol β by DOB suggests that this lesion provides a chemical explanation for the cytotoxicity of drugs that produce it and explains previously unexplained observations in the literature concerning abasic lesions that are not repaired efficiently. Finally, these observations provide the impetus for the design of a new family of inhibitors of Pol β.
Co-reporter:Aaron C. Jacobs ; Marino J. E. Resendiz
Journal of the American Chemical Society 2010 Volume 132(Issue 11) pp:3668-3669
Publication Date(Web):February 25, 2010
DOI:10.1021/ja100281x
RNA oxidation is important in the etiology of disease and as a tool for studying the structure and folding kinetics of this biopolymer. Nucleobase radicals are the major family of reactive intermediates produced in RNA exposed to diffusible species such as hydroxyl radical. The nucleobase radicals are believed to produce direct strand breaks by abstracting hydrogen atoms from their own and neighboring ribose rings. By independently generating the formal C5 hydrogen atom addition product of uridine in RNA, we provide the first chemical characterization of the pathway for direct strand scission from an RNA nucleobase radical. The process is more efficient under anaerobic conditions. The preference for strand scission in double-stranded RNA over single-stranded RNA suggests that this chemistry may be useful for analyzing the secondary structure of RNA in hydroxyl radical cleavage experiments if they are carried out under anaerobic conditions.
Co-reporter:Lirui Guan, Katarzyna Bebenek, Thomas A. Kunkel, and Marc M. Greenberg
Biochemistry 2010 Volume 49(Issue 45) pp:
Publication Date(Web):October 20, 2010
DOI:10.1021/bi101533a
5′-(2-Phosphoryl-1,4-dioxobutane) (DOB) is an oxidized abasic lesion that is produced by a variety of DNA damaging agents, including several antitumor antibiotics. DOB efficiently and irreversibly inhibits DNA polymerase β, an essential base excision repair enzyme in mammalian cells. The generality of this mode of inhibition by DOB is supported by the inactivation of DNA polymerase λ, which may serve as a possible backup for DNA polymerase β during abasic site repair. Protein digests suggest that Lys72 and Lys84, which are present in the lyase active site of DNA polymerase β, are modified by DOB. Monoaldehyde analogues of DOB substantiate the importance of the 1,4-dicarbonyl component of DOB for efficient inactivation of Pol β and the contribution of a freely diffusible electrophile liberated from the inhibitor by the enzyme. Inhibition of DNA polymerase β’s lyase function is accompanied by inactivation of its DNA polymerase activity as well, which prevents long patch base excision repair of DOB. Overall, DOB is highly refractory to short patch and long patch base excision repair. Its recalcitrance to succumb to repair suggests that DOB is a significant source of the cytotoxicity of DNA damaging agents that produce it.
Co-reporter:Xiaohua Peng, Avik K. Ghosh, Bennett Van Houten and Marc M. Greenberg
Biochemistry 2010 Volume 49(Issue 1) pp:
Publication Date(Web):December 9, 2009
DOI:10.1021/bi901603h
The DNA radical resulting from formal abstraction of a hydrogen atom from the thymidine methyl group, 5-(2′-deoxyuridinyl)methyl radical, forms interstrand cross-links with the opposing 2′-deoxyadenosine. This is the first chemically characterized, radical-mediated cross-link between two opposing nucleotides. In addition, cross-linking between opposing bases in the duplex is less common than between those separated by one or two nucleotides. The first step in cross-link repair was investigated using the UvrABC bacterial nucleotide excision repair system. UvrABC incised both strands of the cross-linked DNA, although the strand containing the cross-linked purine was preferred by the enzyme in two different duplexes. The incision sites in one strand were spaced 11−14 nucleotides apart, as is typical for UvrABC incision. The majority of incisions occur at the third phosphate from the 3′-side of the cross-link and eighth or ninth phosphate on the 5′-side. In addition, cleavage was found to occur on both strands, producing double-strand breaks in ∼25−29% of the incision events. This is the first example of double-strand cleavage during nucleotide excision repair of cross-linked DNA that does not already contain a strand break in the vicinity of the cross-link.
Co-reporter:Remus S. Wong, Jonathan T. Sczepanski and Marc M. Greenberg
Chemical Research in Toxicology 2010 Volume 23(Issue 4) pp:766
Publication Date(Web):March 16, 2010
DOI:10.1021/tx9003984
The C2′-oxidized abasic lesion (C2-AP) is produced in DNA that is subjected to oxidative stress. The lesion disrupts replication and gives rise to mutations that are dependent upon the identity of the upstream nucleotide. Ape1 incises C2-AP, but the 5′-phosphorylated fragment is not a substrate for the lyase activity of DNA polymerase β. Excision of the lesion is achieved by strand displacement synthesis in the presence of flap endonuclease during which C2-AP and the 3′-adjacent nucleotide are replaced. The oxidized abasic lesion is also a substrate for the bacterial UvrABC nucleotide excision repair system. These data suggest that the redundant nature of DNA repair systems provides a means for removing a lesion that resists excision by short patch base excision repair.
Co-reporter:Hui Ding and Marc M. Greenberg
The Journal of Organic Chemistry 2010 Volume 75(Issue 3) pp:535-544
Publication Date(Web):January 12, 2010
DOI:10.1021/jo902071y
The 5-halopyrimidine nucleotides damage DNA upon UV-irradiation or exposure to γ-radiolysis via the formation of the 2′-deoxyuridin-5-yl σ-radical. The bromo and iodo derivatives of these molecules are useful tools for probing DNA structure and as therapeutically useful radiosensitizing agents. A series of aryl iodide C-nucleotides were incorporated into synthetic oligonucleotides and exposed to UV-irradiation and γ-radiolysis. The strand damage produced upon irradiation of DNA containing these molecules is consistent with the generation of highly reactive σ-radicals. Direct stand breaks and alkali-labile lesions are formed at the nucleotide analogue and flanking nucleotides. The distribution of lesion type and location varies depending upon the position of the aryl ring that is iodinated. Unlike 5-halopyrimidine nucleotides, the aryl iodides produce interstrand cross-links in duplex regions of DNA when exposed to γ-radiolysis or UV-irradiation. Quenching studies suggest that cross-links are produced by γ-radiolysis via capture of a solvated electron, and subsequent fragmentation to the σ-radical. These observations suggest that aryl iodide C-nucleotide analogues may be useful as probes for excess electron transfer and radiosensitizing agents.
Co-reporter:Remus S. Wong;Jonathan T. Sczepanski;Gregory D. Bowman;Jeffrey N. McKnight
PNAS 2010 Volume 107 (Issue 52 ) pp:22475-22480
Publication Date(Web):2010-12-28
DOI:10.1073/pnas.1012860108
Apurinic/apyrimidinic (AP) sites are ubiquitous DNA lesions that are highly mutagenic and cytotoxic if not repaired. In addition,
clusters of two or more abasic lesions within one to two turns of DNA, a hallmark of ionizing radiation, are repaired much
less efficiently and thus present greater mutagenic potential. Abasic sites are chemically labile, but naked DNA containing
them undergoes strand scission slowly with a half-life on the order of weeks. We find that independently generated AP sites
within nucleosome core particles are highly destabilized, with strand scission occurring ∼60-fold more rapidly than in naked
DNA. The majority of core particles containing single AP lesions accumulate DNA–protein cross-links, which persist following
strand scission. The N-terminal region of histone protein H4 contributes significantly to DNA–protein cross-links and strand
scission when AP sites are produced approximately 1.5 helical turns from the nucleosome dyad, which is a known hot spot for
nucleosomal DNA damage. Reaction rates for AP sites at two positions within this region differ by ∼4-fold. However, the strand
scission of the slowest reacting AP site is accelerated when it is part of a repair resistant bistranded lesion composed of
two AP sites, resulting in rapid formation of double strand breaks in high yields. Multiple lysine residues within a single
H4 protein catalyze double strand cleavage through a mechanism believed to involve a templating effect. These results show
that AP sites within the nucleosome produce significant amounts of DNA–protein cross-links and generate double strand breaks,
the most deleterious form of DNA damage.
Co-reporter:Jonathan T. Sczepanski ; Aaron C. Jacobs ; Ananya Majumdar
Journal of the American Chemical Society 2009 Volume 131(Issue 31) pp:11132-11139
Publication Date(Web):July 14, 2009
DOI:10.1021/ja903404v
The C4′-oxidized abasic site (C4-AP) is a commonly formed DNA lesion, which generates two types of interstrand cross-links (ICLs). The kinetically favored cross-link consists of two full length strands and forms reversibly and exclusively with dA. Cross-link formation is attributed to condensation of C4-AP with the N6-amino group of dA. Formation of the thermodynamic ICL involves cleavage of the strand containing C4-AP on the 3′-side of the lesion. The ratios and yields of the ICLs are highly dependent upon the local sequence. Product analysis of enzyme-digested material reveals that the ICL with dA is a cyclic adduct. Formation of the thermodynamically favored cross-link is catalyzed by the surrounding DNA sequence and occurs favorably with dC and dA but not with dG or dT. Mechanistic studies indicate that β-elimination from C4-AP is the rate-limiting step in the formation of the thermodynamic ICL and that the local DNA environment determines the rate constant for this reaction. The efficiency of ICL formation, the stability of the thermodynamic products, and their possible formation in cells (Regelus, P.; et al. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14032) suggest that these lesions will be deleterious to the biological system in which they are produced.
Co-reporter:Haidong Huang, Shuhei Imoto and Marc M. Greenberg
Biochemistry 2009 Volume 48(Issue 33) pp:
Publication Date(Web):July 20, 2009
DOI:10.1021/bi900927d
Tandem lesions are comprised of two contiguously damaged nucleotides. Tandem lesions make up the major family of reaction products generated from a pyrimidine nucleobase radical, which are formed in large amounts by ionizing radiation. One of these tandem lesions contains a thymidine glycol lesion flanked on its 5′-side by 2-deoxyribonolactone (LTg). The replication of this tandem lesion was investigated in Escherichia coli using single-stranded genomes. LTg is a much more potent replication block than thymidine glycol and is bypassed only under SOS-induced conditions. The adjacent thymidine glycol does not significantly affect nucleotide incorporation opposite 2-deoxyribonolactone in wild-type cells. In contrast, the misinsertion frequency opposite thymidine glycol, which is negligible in the absence of 2-deoxyribonolactone, increases to 10% in wild-type cells when LTg is flanked by a 3′-dG. Experiments in which the flanking nucleotides are varied and in cells lacking one of the SOS-induced bypass polymerases indicate that the mutations are due to a mechanism in which the primer misaligns prior to bypassing the lesion, which allows for an additional nucleotide to be incorporated across from the 3′-flanking nucleotide. Subsequent realignment and extension results in the observed mutations. DNA polymerases II and IV are responsible for misalignment induced mutations and compete with DNA polymerase V which reads through the tandem lesion. These experiments reveal that incorporation of the thymidine glycol into a tandem lesion indirectly induces increases in mutations by blocking replication, which enables the misalignment−realignment mechanism to compete with direct bypass by DNA polymerase V.
Co-reporter:Shuhei Imoto, Leslie A. Bransfield, Deborah L. Croteau, Bennett Van Houten and Marc M. Greenberg
Biochemistry 2008 Volume 47(Issue 14) pp:
Publication Date(Web):March 15, 2008
DOI:10.1021/bi7021427
DNA tandem lesions are comprised of two contiguously damaged nucleotides. This subset of clustered lesions is produced by a variety of oxidizing agents, including ionizing radiation. Clustered lesions can inhibit base excision repair (BER). We report the effects of tandem lesions composed of a thymine glycol and a 5′-adjacent 2-deoxyribonolactone (LTg) or tetrahydrofuran abasic site (FTg). Some BER enzymes that act on the respective isolated lesions do not accept the tandem lesion as a substrate. For instance, endonuclease III (Nth) does not excise thymine glycol (Tg) when it is part of either tandem lesion. Similarly, endonuclease IV (Nfo) does not incise L or F when they are in tandem with Tg. Long-patch BER overcomes inhibition by the tandem lesion. DNA polymerase β (Pol β) carries out strand displacement synthesis, following APE1 incision of the abasic site. Pol β activity is enhanced by flap endonuclease (FEN1), which cleaves the resulting flap. The tandem lesion is also incised by the bacterial nucleotide excision repair system UvrABC with almost the same efficiency as an isolated Tg. These data reveal two solutions that DNA repair systems can use to counteract the formation of tandem lesions.
Co-reporter:Marc M. Greenberg
Organic & Biomolecular Chemistry 2007 vol. 5(Issue 1) pp:18-30
Publication Date(Web):14 Nov 2006
DOI:10.1039/B612729K
DNA damage is a double-edged sword. The modifications produced in the biopolymer are associated with aging, and give rise to a variety of diseases, including cancer. DNA is also the target of anti-tumor agents and the most generally used nonsurgical treatment of cancer, ionizing radiation. Agents that damage DNA produce a variety of radicals. Elucidating the chemistry of individual DNA radicals is challenging due to the availability of multiple reactive pathways and complexities inherent with carrying out mechanistic studies on a heterogeneous polymer. The ability to independently generate radicals and their metastable products at defined sites in DNA has greatly facilitated understanding this biologically important chemistry.
Co-reporter:Hui Ding and Marc M. Greenberg
Chemical Research in Toxicology 2007 Volume 20(Issue 11) pp:1623
Publication Date(Web):October 17, 2007
DOI:10.1021/tx7002307
Interstrand cross-links are minor components of the collection of products formed in DNA by ionizing radiation. Through their formation by other damaging agents, it is known that interstrand cross-links exert significant effects on replication and transcription. The structures of DNA interstrand cross-links produced as a result of γ-radiolysis are unknown. Using synthetic duplexes we found that interstrand cross-link formation required thymidine and occurred with G values of ∼10−4 nmol J−1. Enzymatic digestion of a tritiated substrate indicated that interstrand cross-links were derived from the reaction of 5-(2′-deoxyuridinyl)methyl radical (1) with the opposing 2′-deoxyadenosine to yield 5, which was identical to the product previously characterized when 1 was independently generated from a synthetic precursor. Conservative estimates indicated that 5 accounted for at least one-fourth of the interstrand cross-links produced in DNA by γ-radiolysis. Utilization of a probe designed specifically to detect hole migration suggested that ∼20% of the interstrand cross-links were produced by γ-radiolysis via this pathway. Experiments using an independent source of hydroxyl radical indicated that cross-links were also produced by this species. Hence, DNA interstrand cross-links arising from 1 should result from a variety of oxidative stress mechanisms.
Co-reporter:Liang Xue;Marc M. Greenberg Dr.
Angewandte Chemie International Edition 2007 Volume 46(Issue 4) pp:
Publication Date(Web):8 DEC 2006
DOI:10.1002/anie.200603454
Throwing light on lesions: DNA oxidation is a ubiquitous but potentially dangerous process that produces a variety of structural modifications in the biopolymer. One modified unit, 2-deoxyribonolactone (L), produces cross-links with DNA repair proteins and is mutagenic. Selective fluorescence sensors (e.g. 1) show that L is the major alkali-labile deoxyribose lesion produced in DNA exposed to γ radiolysis.
Co-reporter:Liang Xue;Marc M. Greenberg Dr.
Angewandte Chemie 2007 Volume 119(Issue 4) pp:
Publication Date(Web):8 DEC 2006
DOI:10.1002/ange.200603454
Läsionen erhellt: Die DNA-Oxidation ist ein allgegenwärtiger, jedoch potenziell gefährlicher Prozess, der zu einer Vielzahl an Strukturveränderungen im Biopolymer führt. Eine veränderte Einheit, 2-Desoxyribonolacton (L), erzeugt Vernetzungen mit DNA-Reparaturproteinen und ist mutagen. Mit selektiven Fluoreszenzsensoren (z. B. 1) lässt sich L als hauptsächliche alkalilabile Desoxyriboseläsion identifizieren, die in γ-Strahlung ausgesetzter DNA entsteht.
Co-reporter:Jae-Taeg Hwang, Francis E. Baltasar, Daniel L. Cole, David S. Sigman, Chi-hong B. Chen, Marc M. Greenberg
Bioorganic & Medicinal Chemistry 2003 Volume 11(Issue 10) pp:2321-2328
Publication Date(Web):15 May 2003
DOI:10.1016/S0968-0896(03)00071-3
Inhibition of gene expression was recently achieved by targeting the transcriptionally competent open complex using relatively short, pentameric modified oligonucleotides at ∼60 μM. Corroborative affinity cleavage experiments using the copper complex of a phenanthroline conjugate provided the impetus to synthesize additional analogues containing substituents at the 2′-position of uridine in a derivative of 5′-GUGGA (−4 to +1), with the purpose of inhibiting transcription at lower concentrations. Conjugates of 5′-GUGGA modified at the 2′-position of uridine were convergently synthesized using a recently reported method. Seven analogues based upon the 5′-GUGGA scaffold were tested for their ability to inhibit transcription of the lac UV-5 operon. The conjugate containing a tethered pyrene showed 70% inhibition at 20 μM, and modest inhibition at as low as 5 μM. This is a significant improvement over previously tested pentanucleotides and provides direction for the preparation of a next generation of inhibitors.Graphic
Co-reporter:Jaeseung Kim;Jun Mo Gil Dr.
Angewandte Chemie 2003 Volume 115(Issue 47) pp:
Publication Date(Web):8 DEC 2003
DOI:10.1002/ange.200352102
Aus dem Schaden Nutzen ziehen: Die C4′-Position der Desoxyribose ist der Angriffspunkt vieler DNA-schädigender Verbindungen, darunter Bleomycin. Hauptprodukt nach Wasserstoffabstraktion an dieser Position ist ein Oligonucleotid mit C4′-AP-Stelle. Vorgestellt wird hier eine allgemeine Synthesemethode für Oligonucleotide 1 mit C4′-AP-Stellen an definierten Positionen (siehe Schema). Diese Methode sollte physikochemische, biochemische und biologische Untersuchungen dieser häufig auftretenden DNA-Schädigung erleichtern.
Co-reporter:Jaeseung Kim;Jun Mo Gil Dr.
Angewandte Chemie International Edition 2003 Volume 42(Issue 47) pp:
Publication Date(Web):8 DEC 2003
DOI:10.1002/anie.200352102
Selective introduction of damage: The C4′-position of DNA serves as the site of attack for a number of DNA damaging agents, including bleomycin. The C4′-abasic site is a major product found in damaged DNA following abstraction of the hydrogen atom from this position. The first general method for the synthesis of oligonucleotides 1 containing the C4′-abasic site at defined positions is described (see scheme). This method should facilitate physicochemical, biochemical, and biological studies on this commonly formed DNA lesion.
Co-reporter:Marc M. Greenberg
Organic & Biomolecular Chemistry 2007 - vol. 5(Issue 1) pp:NaN30-30
Publication Date(Web):2006/11/14
DOI:10.1039/B612729K
DNA damage is a double-edged sword. The modifications produced in the biopolymer are associated with aging, and give rise to a variety of diseases, including cancer. DNA is also the target of anti-tumor agents and the most generally used nonsurgical treatment of cancer, ionizing radiation. Agents that damage DNA produce a variety of radicals. Elucidating the chemistry of individual DNA radicals is challenging due to the availability of multiple reactive pathways and complexities inherent with carrying out mechanistic studies on a heterogeneous polymer. The ability to independently generate radicals and their metastable products at defined sites in DNA has greatly facilitated understanding this biologically important chemistry.
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