Maintaining the chemical integrity of DNA in the face of assault by oxidizing agents is a constant challenge for living organisms. Base-excision repair has an important role in preventing mutations associated with a common product of oxidative damage to DNA, 8-oxoguanine. Recent structural studies have shown that 8-oxoguanine DNA glycosylases use an intricate series of steps to locate and excise 8-oxoguanine lesions efficiently against a high background of undamaged bases. The importance of preventing mutations associated with 8-oxoguanine is shown by a direct association between defects in the DNA glycosylase MUTYH and colorectal cancer. The properties of other guanine oxidation products and the associated DNA glycosylases that remove them are now also being revealed.

MUTYH-associated polyposis (MAP) is the only inherited colorectal cancer syndrome that is associated with inherited biallelic mutations in a base excision repair gene. The MUTYH glycosylase plays an important role in preventing mutations associated with 8-oxoguanine (OG) by removing adenine residues that have been misincorporated opposite OG. MAP-associated mutations are present throughout MUTYH, with a large number coding for missense variations. To date the available information on the functional properties of MUTYH variants is conflicting. In this study, a kinetic analysis of the adenine glycosylase activity of MUTYH and several variants was undertaken using a correction for active fraction to control for differences due to overexpression and purification. Using these methods, the rate constants for steps involved in the adenine removal process were determined for the MAP variants Y165C, G382D, P391L and Q324R MUTYH. Under single-turnover conditions, the rate of adenine removal for these four variants was found to be 30-40% of WT MUTYH. In addition, the ability of MUTYH and the variants to suppress mutations and complement for the absence of MutY in E. coli was assessed using rifampicin resistance assays. The presence of WT and Q324R MUTYH resulted in complete suppression of the mutation frequency, while G382D MUTYH showed reduced ability to suppress the mutation frequency. In contrast, the mutation frequency observed upon expression of P391L and Y165C MUTYH were similar to the controls, suggesting no activity toward preventing DNA mutations. Notably, though all variations studied herein resulted in similar reductions in adenine glycosylase activity, the effects in the bacterial complementation are quite different. This suggests that the consequences of a specific amino acid variation on overall repair in a cellular context may be magnified.

Biallelic germline mutations in the base excision repair enzyme gene MUTYH lead to multiple colorectal adenomas and carcinomas referred to as MUTYH-associated polyposis. MUTYH removes adenine misincorporated opposite the DNA oxidation product, 8-oxoguanine (OG), thereby preventing accumulation of G:C to T:A transversion mutations. The most common cancer-associated MUTYH variant proteins when expressed in bacteria exhibit reduced OG:A mismatch affinity and adenine removal activity. However, direct evaluation of OG:A mismatch repair efficiency in mammalian cells has not been assessed due to the lack of an appropriate assay. To address this, we developed a novel fluorescence-based assay of OG:A repair and measured the repair capacity of MUTYH-associated polyposis variants expressed in Mutyh-/- mouse embryonic fibroblasts (MEFs). The repair of a single site-specific synthetic lesion in a green fluorescent protein reporter leads to green fluorescent protein expression with co-expression of a red fluorescent protein serving as the transfection control. Cell lines that stably express the MUTYH-associated polyposis variants G382D and Y165C have significantly lower OG:A repair versus wild-type MEFs and MEFs expressing human wild-type MUTYH. The MUTYH allele that encodes the Q324H variant is found at a frequency above 40% in samples from different ethnic groups and has long been considered phenotypically silent but has recently been associated with increased cancer risk in several clinical studies. In vitro analysis of Q324H MUTYH expressed in insect cells showed that it has reduced enzyme activity similar to that of the known cancer variant G382D. Moreover, we find that OG:A repair in MEFs expressing Q324H was significantly lower than wild-type controls, establishing that Q324H is functionally impaired and providing further evidence that this common variant may lead to increased cancer risk.

The oxidation product of 2'-deoxyguanosine, 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG), produces G:C to T:A transversion mutations. The Escherichia coli base excision repair glycosylase MutY plays an important role in preventing OG-associated mutations by removing adenines misincorporated opposite OG lesions during DNA replication. Recently, biallelic mutations in the human MutY homologue (hMYH) have been correlated with the development of colorectal cancer. The two most common mutations correspond to two single amino acid substitutions in the hMYH protein: Y165C and G382D [Al-Tassan, N., et al. (2002) Nat. Genet. 30, 227-232]. Previously, our laboratory analyzed the adenine glycosylase activity of the homologous variant E. coli MutY enzymes, Y82C and G253D [Chmiel, N. H., et al. (2003) J. Mol. Biol. 327, 431-443]. This work demonstrated that both variants have a reduced adenine glycosylase activity and affinity for substrate analogues compared to wild-type MutY. Recent structural work on Bacillus stearothermophilus MutY bound to an OG:A mismatch-containing duplex indicates that both residues aid in recognition of OG [Fromme, J. C., et al. (2004) Nature 427, 652-656]. To determine the extent with which Tyr 82 and Gly 253 contribute to catalysis of adenine removal by E. coli MutY, we made a series of additional modifications in these residues, namely, Y82F, Y82L, and G253A. When the substrate analogue 2'-deoxy-2'-fluoroadenosine (FA) in duplex paired with G or OG is used, both Y82F and G253A showed reduced binding affinity, and G253A was unable to discriminate between OG and G when paired with FA. Additionally, compromised glycosylase activity of Y82F, Y82C, and G253A MutY was observed using the nonoptimal G:A substrate, or at low reaction temperatures. Interestingly, adenine removal from an OG:A-containing DNA substrate by Y82C MutY was also shown to be extremely sensitive to the NaCl concentration. The most surprising result was the remarkably similar activity of Y82L MutY to the WT enzyme under all conditions examined, indicating that a leucine residue may effectively replace tyrosine for intercalation at the OG:A mismatch. The results contained herein provide further insight regarding the intricate roles of Tyr 82 and Gly 253 in the OG recognition and adenine excision functions of MutY.

MUTYH-associated polyposis (MAP) is a colorectal cancer predisposition syndrome that is caused by inherited biallelic mutations in the base excision repair (BER) gene, MUTYH. MUTYH is a DNA glycosylase that removes adenine (A) misinserted opposite 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG). In this work, wild type (WT) MUTYH overexpressed using a baculovirus-driven insect cell expression system (BEVS) provided significantly higher levels of enzyme compared to bacterial overexpression. The isolated MUTYH enzyme was analyzed for potential post-translational modifications using mass spectrometry. An in vivo phosphorylation site was validated at Serine 524, which is located in the C-terminal OG recognition domain within the proliferating cell nuclear antigen (PCNA) binding region. Characterization of the phosphomimetic (S524D) and phosphoablating (S524A) mutants together with the observation that Ser 524 can be phosphorylated suggest that this residue may play an important regulatory role in vivo by altering stability and OG:A mismatch affinity.