Sheila S. David

Inherited variants of MYH associated with somatic G:C→ T:A mutations in colorectal tumors

Inherited defects of base excision repair have not been associated with any human genetic disorder, although mutations of the genes mutM and mutY, which function in Escherichia coli base excision repair, lead to increased transversions of G:C to T:A. We have studied family N, which is affected with multiple colorectal adenomas and carcinoma but lacks an inherited mutation of the adenomatous polyposis coli gene (APC) that is associated with familial adenomatous polyposis. Here we show that 11 tumors from 3 affected siblings contain 18 somatic inactivating mutations of APC and that 15 of these mutations are G:C→T:A transversions—a significantly greater proportion than is found in sporadic tumors or in tumors associated with familial adenomatous polyposis. Analysis of the human homolog of mutY, MYH, showed that the siblings were compound heterozygotes for the nonconservative missense variants Tyr165Cys and Gly382Asp. These mutations affect residues that are conserved in mutY of E. coli (Tyr82 and Gly253). Tyrosine 82 is located in the pseudo-helix-hairpin-helix (HhH) motif and is predicted to function in mismatch specificity. Assays of adenine glycosylase activity of the Tyr82Cys and Gly253Asp mutant proteins with 8-oxoG:A and G:A substrates show that their activity is reduced significantly. Our findings link the inherited variants in MYH to the pattern of somatic APC mutation in family N and implicate defective base excision repair in predisposition to tumors in humans.

Base Excision Repair of Oxidative DNA Damage

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.

DNA-mediated charge transport for DNA repair

MutY, like many DNA base excision repair enzymes, contains a [4Fe4S]2+ cluster of undetermined function. Electrochemical studies of MutY bound to a DNA-modified gold electrode demonstrate that the [4Fe4S] cluster of MutY can be accessed in a DNA-mediated redox reaction. Although not detectable without DNA, the redox potential of DNA-bound MutY is ≈275 mV versus NHE, which is characteristic of HiPiP iron proteins. Binding to DNA is thus associated with a change in [4Fe4S]3+/2+ potential, activating the cluster toward oxidation. Given that DNA charge transport chemistry is exquisitely sensitive to perturbations in base pair structure, such as mismatches, we propose that this redox process of MutY bound to DNA exploits DNA charge transport and provides a DNA signaling mechanism to scan for mismatches and lesions in vivo.

Superior removal of hydantoin lesions relative to other oxidized bases by the human DNA glycosylase hNEIL1.

The DNA glycosylase hNEIL1 initiates the base excision repair (BER) of a diverse array of lesions, including ring-opened purines and saturated pyrimidines. Of these, the hydantoin lesions, guanidinohydantoin (Gh) and the two diastereomers of spiroiminodihydantoin (Sp1 and Sp2), have garnered much recent attention due to their unusual structures, high mutagenic potential, and detection in cells. In order to provide insight into the role of repair, the excision efficiency by hNEIL1 of these hydantoin lesions relative to other known substrates was determined. Most notably, quantitative examination of the substrate specificity with hNEIL1 revealed that the hydantoin lesions are excised much more efficiently (>100-fold faster) than the reported standard substrates thymine glycol (Tg) and 5-hydroxycytosine (5-OHC). Importantly, the glycosylase and beta,delta-lyase reactions are tightly coupled such that the rate of the lyase activity does not influence the observed substrate specificity. The activity of hNEIL1 is also influenced by the base pair partner of the lesion, with both Gh and Sp removal being more efficient when paired with T, G, or C than when paired with A. Notably, the most efficient removal is observed with the Gh or Sp paired in the unlikely physiological context with T; indeed, this may be a consequence of the unstable nature of base pairs with T. However, the facile removal via BER in promutagenic base pairs that are reasonably formed after replication (such as Gh.G) may be a factor that modulates the mutagenic profile of these lesions. In addition, hNEIL1 excises Sp1 faster than Sp2, indicating the enzyme can discriminate between the two diastereomers. This is the first time that a BER glycosylase has been shown to be able to preferentially excise one diastereomer of Sp. This may be a consequence of the architecture of the active site of hNEIL1 and the structural uniqueness of the Sp lesion. These results indicate that the hydantoin lesions are the best substrates identified thus far for hNEIL1 and suggest that repair of these lesions may be a critical function of the hNEIL1 enzyme in vivo.

RNA editing changes the lesion specificity for the DNA repair enzyme NEIL1

Editing of the pre-mRNA for the DNA repair enzyme NEIL1 causes a lysine to arginine change in the lesion recognition loop of the protein. The two forms of NEIL1 are shown here to have distinct enzymatic properties. The edited form removes thymine glycol from duplex DNA 30 times more slowly than the form encoded in the genome, whereas editing enhances repair of the guanidinohydantoin lesion by NEIL1. In addition, we show that the NEIL1 recoding site is a preferred editing site for the RNA editing adenosine deaminase ADAR1. The edited adenosine resides in an A-C mismatch in a hairpin stem formed by pairing of exon 6 to the immediate upstream intron 5 sequence. As expected for an ADAR1 site, editing at this position is increased in human cells treated with interferon α. These results suggest a unique regulatory mechanism for DNA repair and extend our understanding of the impact of RNA editing.

Mutation versus repair: NEIL1 removal of hydantoin lesions in single-stranded, bulge, bubble, and duplex DNA contexts.

Human DNA glycosylase NEIL1 exhibits a superior ability to remove oxidized guanine lesions guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) from duplex DNA in comparison to other substrates. In this work, Gh and Sp lesions in bubble, bulge, and single-stranded DNA were found to be good substrates for NEIL1 but were typically excised at much slower rates than from canonical duplex substrates. A notable exception was the activity of NEIL1 on removal of Gh in bubble structures which approaches that of the normal duplex substrate. The cleavage of Gh in the template strand of a replication or transcription bubble may prevent mutations associated with Gh during replication or transcription. However, removal of hydantoin lesions in the absence of an opposite base may also result in strand breaks and potentially deletion and frameshift mutations. Consistent with this as a potential mechanism leading to an N-1 frameshift mutation, the nick left after the removal of the Gh lesion in a DNA bulge by NEIL1 was efficiently religated in the presence of polynucleotide kinase (PNK) and human DNA ligase III (Lig III). These results indicate that NEIL1 does not require a base opposite to identify and remove hydantoin lesions. Depending on the context, the glycosylase activity of NEIL1 may stall replication and prevent mutations or lead to inappropriate removal that may contribute to the mutational spectrum of these unusual lesions.

Direct Fluorescence Monitoring of DNA Base Excision Repair

Uracil is an undesired component of DNA, as it arises from spontaneous deamination of cytosine.[1] This hydrolysis reaction promotes mutations, since the resulting U-G pair can be misread during DNA replication. As a result, multiple cellular enzymes have evolved to detect uracil in DNA and remove it prior to replication.[2] In E. coli uracil DNA glycosylase (UDG) enzyme functions to guard the bacterial genome. In humans, similar enzyme activities exist, including the proteins UNG1/2, SMUG, and TDG.[3] These enzymes flip uracil out of the DNA helix and cleave it from its deoxyribose sugar, leaving an abasic site in its place.[4]

Adenine removal activity and bacterial complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer.

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 Escherichia 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.

Surprising repair activities of nonpolar analogs of 8-oxoG expose features of recognition and catalysis by base excision repair glycosylases.

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.

Cancer-associated variants and a common polymorphism of MUTYH exhibit reduced repair of oxidative DNA damage using a GFP-based assay in mammalian cells

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.