S K-edge X-ray absorption spectroscopy (XAS) was used to study the [Fe4S4] clusters in the DNA repair glycosylases EndoIII and MutY to evaluate the effects of DNA binding and solvation on Fe–S bond covalencies (i.e., the amount of S 3p character mixed into the Fe 3d valence orbitals). Increased covalencies in both iron–thiolate and iron–sulfide bonds would stabilize the oxidized state of the [Fe4S4] clusters. The results are compared to those on previously studied [Fe4S4] model complexes, ferredoxin (Fd), and to new data on high-potential iron–sulfur protein (HiPIP). A limited decrease in covalency is observed upon removal of solvent water from EndoIII and MutY, opposite to the significant increase observed for Fd, where the [Fe4S4] cluster is solvent exposed. Importantly, in EndoIII and MutY, a large increase in covalency is observed upon DNA binding, which is due to the effect of its negative charge on the iron–sulfur bonds. In EndoIII, this change in covalency can be quantified and makes a significant contribution to the observed decrease in reduction potential found experimentally in DNA repair proteins, enabling their HiPIP-like redox behavior.

•MutY is a unique BER glycosylase that removes adenine from 8-oxoG:A mispairs.•Dysfunctional MUTYH variants are associated with MUTYH-associated polyposis (MAP).•There are many MAP variants that have a myriad of functional consequences.•MUTYH contains a recently identified Zinc-linchpin motif that is required for efficient mismatch recognition and repair.•New structural and mechanistic information indicated that MutY is a retaining glycosidase.Reactive oxygen and nitrogen species (RONS) may infringe on the passing of pristine genetic information by inducing DNA inter- and intra-strand crosslinks, protein-DNA crosslinks, and chemical alterations to the sugar or base moieties of DNA. 8-Oxo-7,8-dihydroguanine (8-oxoG) is one of the most prevalent DNA lesions formed by RONS and is repaired through the base excision repair (BER) pathway involving the DNA repair glycosylases OGG1 and MUTYH in eukaryotes. MUTYH removes adenine (A) from 8-oxoG:A mispairs, thus mitigating the potential of G:C to T:A transversion mutations from occurring in the genome. The paramount role of MUTYH in guarding the genome is well established in the etiology of a colorectal cancer predisposition syndrome involving variants of MUTYH, referred to as MUTYH-associated polyposis (MAP). In this review, we highlight recent advances in understanding how MUTYH structure and related function participate in the manifestation of human disease such as MAP. Here we focus on the importance of MUTYH's metal cofactor sites, including a recently discovered “Zinc linchpin” motif, as well as updates to the catalytic mechanism. Finally, we touch on the insight gleaned from studies with MAP-associated MUTYH variants and recent advances in understanding the multifaceted roles of MUTYH in the cell, both in the prevention of mutagenesis and tumorigenesis.Download high-res image (199KB)Download full-size image

Aberrant epigenetic methylation is linked to the onset and progression of cancer. In this issue of Chemistry & Biology, Furst and Barton (2015) describe a sensitive electrochemical assay that can detect hyperactive epigenetic methylation in tumor tissue.

Mammalian MutY glycosylases have a unique architecture that features an interdomain connector (IDC) that joins the catalytic N-terminal domain and 8-oxoguanine (OG) recognition C-terminal domain. The IDC has been shown to be a hub for interactions with protein partners involved in coordinating downstream repair events and signaling apoptosis. Herein, a previously unidentified zinc ion and its coordination by three Cys residues of the IDC region of eukaryotic MutY organisms were characterized by mutagenesis, ICP-MS, and EXAFS. In vitro kinetics and cellular assays on WT and Cys to Ser mutants have revealed an important function for zinc coordination on overall protein stability, iron–sulfur cluster insertion, and ability to mediate DNA damage repair. We propose that this “zinc linchpin” motif serves to structurally organize the IDC and coordinate the damage recognition and base excision functions of the C- and N-terminal domains.

An important feature of the common DNA oxidation product 8-oxo-7,8-dihydroguanine (OG) is its susceptibility to further oxidation that produces guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) lesions. In the presence of amines, G or OG oxidation produces hydantoin amine adducts. Such adducts may form in cells via interception of oxidized intermediates by protein-derived nucleophiles or naturally occurring amines that are tightly associated with DNA. Gh and Sp are known to be substrates for base excision repair (BER) glycosylases; however, large Sp–amine adducts would be expected to be more readily repaired by nucleotide excision repair (NER). A series of Sp adducts differing in the size of the attached amine were synthesized to evaluate the relative processing by NER and BER. The UvrABC complex excised Gh, Sp, and the Sp–amine adducts from duplex DNA, with the greatest efficiency for the largest Sp–amine adducts. The affinity of UvrA for all of the lesion duplexes was found to be similar, whereas the efficiency of UvrB loading tracked with the efficiency of UvrABC excision. In contrast, the human BER glycosylase NEIL1 exhibited robust activity for all Sp–amine adducts irrespective of size. These studies suggest that both NER and BER pathways mediate repair of a diverse set of hydantoin lesions in cells.

The gas-phase thermochemical properties (tautomeric energies, acidity, and proton affinity) have been measured and calculated for adenine and six adenine analogues that were designed to test features of the catalytic mechanism used by the adenine glycosylase MutY. The gas-phase intrinsic properties are correlated to possible excision mechanisms and MutY excision rates to gain insight into the MutY mechanism. The data support a mechanism involving protonation at N7 and hydrogen bonding to N3 of adenine. We also explored the acid-catalyzed (non-enzymatic) depurination of these substrates, which appears to follow a different mechanism than that employed by MutY, which we elucidate using calculations.

MutY prevent DNA mutations associated with 8-oxoguanine (OG) by catalyzing the removal of adenines opposite OG. pH dependence of the adenine glycosylase activity establish that Asp 138 of MutY must be deprotonated for maximal activity consistent with its role in stabilizing the oxacarbenium ion transition state in an SN1 mechanism. A cellular OG:A repair assay allowed further validation of the critical role of Asp 138. Conservative substitutions of the catalytic residues Asp 138 and Glu 37 resulted in enzymes with a range of activity that were used to correlate the efficiency of adenine excision with overall OG:A repair and suppression of DNA mutations in vivo. The results show that MutY variations that exhibit reduced mismatch affinity result in more dramatic reductions in cellular OG:A repair than those that only compromise adenine excision catalysis.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (184 K)Download as PowerPoint slideHighlights► Asp 138 must be deprotonated to stabilize the transition state for maximal activity ► Consequences of altering catalytic amino acids in MutY on cellular mismatch repair ► Correlation of MutY enzymatic parameters with ability to mediate cellular repair ► Importance of high affinity binding of MutY to the OG:A mismatch for high levels of repair

The family 4 uracil–DNA glycosylase from the hyperthermophilic organism Archaeoglobus fulgidus (AFUDG) is responsible for the removal of uracil in DNA as the first step in the base excision repair (BER) pathway. AFUDG contains a large solvent-exposed peptide region containing an α helix and loop anchored on each end via ligation of two cysteine thiolates to a [4Fe–4S]2+ cluster. We propose that this region plays a similar role in DNA damage recognition as a smaller iron–sulfur cluster loop (FCL) motif in the structurally unrelated BER glycosylases MutY and Endonuclease III and therefore refer to this region as the “pseudo-FCL” in AFUDG. In order to evaluate the importance of this region, three positively charged residues (Arg 86, Arg 91, Lys 100) and the anchoring Cys residues (Cys 85, Cys 101) within this motif were replaced with alanine, and the effects of these replacements on uracil excision in single- and double-stranded DNA were evaluated. These results show that this region participates and allows for efficient recognition and excision of uracil within DNA. Notably, R86A AFUDG exhibited reduced activity for uracil removal only within double-stranded DNA, suggesting an importance in duplex disruption and extrusion of the base as part of the excision process. In addition, mutation of the [4Fe–4S]2+ cluster cysteine ligands at the ends of the pseudo-FCL to alanine reduced the uracil excision efficiency, suggesting the importance of anchoring the loop via coordination to the cluster. In contrast, K100A AFUDG exhibited enhanced uracil excision activity, providing evidence for the importance of the loop conformation and flexibility. Taken together, the results herein provide evidence that the pseudo-FCL motif is involved in DNA binding and catalysis, particularly in duplex DNA contexts. This work underscores the requirement of an ensemble of interactions, both distant and in proximity to the damaged site, for accurate and efficient uracil excision.

Repair glycosylases locate and excise damaged bases from DNA, playing central roles in preservation of the genome and prevention of disease. Two key glycosylases, Fpg and hOGG1, function to remove the mutagenic oxidized base 8-oxoG (OG) from DNA. To investigate the relative contributions of conformational preferences, leaving group ability, enzyme-base hydrogen bonding, and nucleobase shape on damage recognition by these glycosylases, a series of four substituted indole nucleosides, based on the parent OG nonpolar isostere 2Cl-4F-indole, were tested as possible direct substrates of these enzymes in the context of 30 base pair duplexes paired with C. Surprisingly, single-turnover experiments revealed that Fpg-catalyzed base removal activity of two of the nonpolar analogs was superior to the native OG substrate. The hOGG1 glycosylase was also found to catalyze removal of three of the nonpolar analogs, albeit considerably less efficiently than removal of OG. Of note, the analog that was completely resistant to hOGG1-catalyzed excision has a chloro-substituent at the position of NH7 of OG, implicating the importance of recognition of this position in catalysis. Both hOGG1 and Fpg retained high affinity for the duplexes containing the nonpolar isosteres. These studies show that hydrogen bonds between base and enzyme are not needed for efficient damage recognition and repair by Fpg and underscore the importance of facile extrusion from the helix in its damaged base selection. In contrast, damage removal by hOGG1 is sensitive to both hydrogen bonding groups and nucleobase shape. The relative rates of excision of the analogs with the two glycosylases highlight key differences in their mechanisms of damaged base recognition and removal.

Two base excision repair glycosylase (BER) transition state (TS) mimics, (3R,4R)-1-benzyl (hydroxymethyl) pyrrolidin-3-ol (1NBn) and (3R,4R)-(hydroxymethyl) pyrrolidin-3-ol (1N), were synthesized using an improved method. Several BER glycosylases that repair oxidized DNA bases, bacterial formamidopyrimdine glycosylase (Fpg), human OG glycosylase (hOGG1) and human Nei-like glycosylase 1 (hNEIL1) exhibit exceptionally high affinity (Kd∼pM) with DNA duplexes containing the 1NBn and 1N nucleotide. Notably, comparison of the Kd values of both TS mimics relative to an abasic analog (THF) in duplex contexts paired opposite C or A suggest that these DNA repair enzymes use distinctly different mechanisms for damaged base recognition and catalysis despite having overlapping substrate specificities.

•We provide a detailed analysis of the glycosylase activity and binding affinity of five MUTYH variants.•A variant in the FCL motif of MUTYH (P281L) has minimal glycosylase activity due to an inability to bind to substrate DNA.•R295C and R520Q MUTYH have low levels of active enzyme, exhibit compromised affinity for damaged DNA and reduced base excision catalysis.•Q324H and P502L have nearly WT glycosylase activity, but exhibit reduced affinity for protein partners Hus1 (of the 9#3−3#1 complex) and PCNA, respectively.•R520Q MUTYH also exhibits compromised affinity for PCNA suggesting a dual role for Arg 520 in OG:A mismatch recognition and interaction with PCNA.MUTYH is a base excision repair (BER) enzyme that prevents mutations in DNA associated with 8-oxoguanine (OG) by catalyzing the removal of adenine from inappropriately formed OG:A base-pairs. Germline mutations in the MUTYH gene are linked to colorectal polyposis and a high risk of colorectal cancer, a syndrome referred to as MUTYH-associated polyposis (MAP). There are over 300 different MUTYH mutations associated with MAP and a large fraction of these gene changes code for missense MUTYH variants. Herein, the adenine glycosylase activity, mismatch recognition properties, and interaction with relevant protein partners of human MUTYH and five MAP variants (R295C, P281L, Q324H, P502L, and R520Q) were examined. P281L MUTYH was found to be severely compromised both in DNA binding and base excision activity, consistent with the location of this variation in the iron-sulfur cluster (FCL) DNA binding motif of MUTYH. Both R295C and R520Q MUTYH were found to have low fractions of active enzyme, compromised affinity for damaged DNA, and reduced rates for adenine excision. In contrast, both Q324H and P502L MUTYH function relatively similarly to WT MUTYH in both binding and glycosylase assays. However, P502L and R520Q exhibited reduced affinity for PCNA (proliferation cell nuclear antigen), consistent with their location in the PCNA-binding motif of MUTYH. Whereas, only Q324H, and not R295C, was found to have reduced affinity for Hus1 of the Rad9–Hus1–Rad1 complex, despite both being localized to the same region implicated for interaction with Hus1. These results underscore the diversity of functional consequences due to MUTYH variants that may impact the progression of MAP.Download high-res image (166KB)Download full-size image