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.     

Structure of the mammalian adenine DNA glycosylase MUTYH: insights into the base excision repair pathway and cancer

Mammalian MutY homologue (MUTYH) is an adenine DNA glycosylase that excises adenine inserted opposite 8-oxoguanine (8-oxoG). The inherited variations in human MUTYH gene are known to cause MUTYH-associated polyposis (MAP), which is associated with colorectal cancer. MUTYH is involved in base excision repair (BER) with proliferating cell nuclear antigen (PCNA) in DNA replication, which is unique and critical for effective mutation-avoidance. It is also reported that MUTYH has a Zn-binding motif in a unique interdomain connector (IDC) region, which interacts with Rad9–Rad1–Hus1 complex (9–1–1) in DNA damage response, and with apurinic/apyrimidinic endonuclease 1 (APE1) in BER. However, the structural basis for the BER pathway by MUTYH and its interacting proteins is unclear. Here, we determined the crystal structures of complexes between mouse MUTYH and DNA, and between the C-terminal domain of mouse MUTYH and human PCNA. The structures elucidated the repair mechanism for the A:8-oxoG mispair including DNA replication-coupled repair process involving MUTYH and PCNA. The Zn-binding motif was revealed to comprise one histidine and three cysteine residues. The IDC, including the Zn-binding motif, is exposed on the MUTYH surface, suggesting its interaction modes with 9–1–1 and APE1, respectively. The structure of MUTYH explains how MAP mutations perturb MUTYH function.     

Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis

The MUTYH DNA glycosylase specifically removes adenine misincorporated by replicative polymerases opposite the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoG). A defective protein activity results in the accumulation of G > T transversions because of unrepaired 8-oxoG:A mismatches. In humans, MUTYH germline mutations are associated with a recessive form of familial adenomatous polyposis and colorectal cancer predisposition (MUTYH-associated polyposis, MAP). Here we studied the repair capacity of the MUTYH variants R171W, E466del, 137insIW, Y165C and G382D, identified in MAP patients. Following expression and purification of human proteins from a bacterial system, we investigated MUTYH incision capacity on an 8-oxoG:A substrate by standard glycosylase assays. For the first time, we employed the surface plasmon resonance (SPR) technology for real-time recording of the association/dissociation of wild-type and MUTYH variants from an 8-oxoG:A DNA substrate. When compared to the wild-type protein, R171W, E466del and Y165C variants showed a severe reduction in the binding affinity towards the substrate, while 137insIW and G382D mutants manifested only a slight decrease mainly due to a slower rate of association. This reduced binding was always associated with impairment of glycosylase activity, with adenine removal being totally abrogated in R171W, E466del and Y165C and only partially reduced in 137insIW and G382D. Our findings demonstrate that SPR analysis is suitable to identify defective enzymatic behaviour even when mutant proteins display minor alterations in substrate recognition.     

Ser 524 is a phosphorylation site in MUTYH and Ser 524 mutations alter 8-oxoguanine (OG): A mismatch recognition

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.     

Interaction of apurinic/apyrimidinic endonuclease 2 (Apn2) with Myh1 DNA glycosylase in fission yeast

Oxidative DNA damage is repaired primarily by the base excision repair (BER) pathway in a process initiated by removal of base lesions or mismatched bases by DNA glycosylases. MutY homolog (MYH, MUTYH, or Myh1) is a DNA glycosylase which excises adenine paired with the oxidative lesion 8-oxo-7,8-dihydroguanine (8-oxoG, or G°), thus reducing G:C to T:A mutations. The resulting apurinic/apyrimidinic (AP) site is processed by an AP-endonuclease or a bifunctional glycosylase/lyase. We show here that the major Schizosaccharomyces pombe AP endonuclease, Apn2, binds to the inter-domain connector located between the N- and C-terminal domains of Myh1. This Myh1 inter-domain connector also interacts with the Hus1 subunit of the Rad9–Rad1–Hus1 checkpoint clamp. Mutagenesis studies indicate that Apn2 and Hus1 bind overlapping but different sequence motifs on Myh1. Mutation on I²⁶¹ of Myh1 reduces its interaction with Hus1, but only slightly attenuates its interaction with Apn2. However, E²⁶² of Myh1 is a key determinant for both Apn2 and Hus1 interactions. Like human APE1, Apn2 has 3′-phosphodiesterase activity. However, unlike hAPE1, Apn2 has a weak AP endonuclease activity which cleaves the AP sites generated by Myh1 glycosylase. Functionally, Apn2 stimulates Myh1 glycosylase activity and Apn2 phosphodiesterase activity is stimulated by Myh1. The cross stimulation of Myh1 and Apn2 enzymatic activities is dependent on their physical interaction. Thus, Myh1 and Apn2 constitute an initial BER complex.     

When you’re strange: Unusual features of the MUTYH glycosylase and implications in cancer

MUTYH is a base-excision repair glycosylase that removes adenine opposite 8-oxoguanine (OG). Variants of MUTYH defective in functional activity lead to MUTYH-associated polyposis (MAP), which progresses to cancer with very high penetrance. Whole genome and whole exome sequencing studies have found MUTYH deficiencies in an increasing number of cancer types. While the canonical OG:A repair activity of MUTYH is well characterized and similar to bacterial MutY, here we review more recent evidence that MUTYH has activities independent of OG:A repair and appear centered on the interdomain connector (IDC) region of MUTYH. We summarize evidence that MUTYH is involved in rapid DNA damage response (DDR) signaling, including PARP activation, 9-1-1 and ATR signaling, and SIRT6 activity. MUTYH alters survival and DDR to a wide variety of DNA damaging agents in a time course that is not consistent with the formation of OG:A mispairs. Studies that suggest MUTYH inhibits the repair of alkyl-DNA damage and cyclopyrimidine dimers (CPDs) is reviewed, and evidence of a synthetic lethal interaction with mismatch repair (MMR) is summarized. Based on these studies we suggest that MUTYH has evolved from an OG:A mispair glycosylase to a multifunctional scaffold for DNA damage response signaling.     

Insight into the roles of tyrosine 82 and glycine 253 in the Escherichia coli adenine glycosylase MutY

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.     

Association of the Rad9–Rad1–Hus1 checkpoint clamp with MYH DNA glycosylase and DNA

Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9–Rad1–Hus1 (9–1–1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9–1–1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9–1–1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134–155) is a key determinant of MYH binding. Both the N-(residues 1–146) and C-terminal (residues 147–280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1^K136A mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad9¹⁻²⁶⁶ (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 9¹⁻²⁶⁶–1–1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 9¹⁻²⁶⁶–1–1 to 5′-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9–1–1 subunits to MYH-directed BER based on subunit asymmetry in protein–protein interactions and DNA binding events.     

MUTYH-associated polyposis--from defect in base excision repair to clinical genetic testing

Established predisposition genes account for only a small proportion of familial colorectal cancer. Recently, it has been shown that germline mutations in MUTYH predispose to MUTYH-associated polyposis (MAP), an autosomal recessive disorder characterised by multiple colorectal adenomas and carcinomas. MUTYH functions as a base excision repair DNA glycosylase that excises adenines misincorporated opposite 8-oxo-7,8-dihydro-2'-deoxyguanosine, one of the most stable products of oxidative DNA damage. It is the failure to correct this mispair that is thought to give rise to the characteristic signature of G:C-->T:A mutations found in MAP-associated tumours. Here, we review the germline mutation spectrum at the MUTYH locus (comprising 30 truncating and 55 missense/inframe insertion/deletion variants) and the molecular mechanism and biochemical defect(s) underlying this disorder. We also discuss the application of molecular genetic analysis of MUTYH in clinical practice.     

Insight into the functional consequences of hMYH variants associated with colorectal cancer: distinct differences in the adenine glycosylase activity and the response to AP endonucleases of Y150C and G365D murine MYH

Escherichia coli MutY and its eukaryotic homologues play an important role in preventing mutations by removing adenine from 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A mismatches. It has recently been demonstrated that inherited biallelic mutations in the genes encoding the human homologue of MutY (hMYH) are correlated with a genetic predisposition for multiple colorectal adenomas and carcinomas. The two most common hMYH variants found in patients with colorectal cancer are Y165C and G382D. In this study, we examined the equivalent variants in the murine MutY homologue (mMYH), Y150C and G365D. The Y150C mMYH enzyme showed a large decrease in the rate of adenine removal from both OG:A- and G:A-containing substrates, while G365D mMYH showed a decrease in the ability to catalyze adenine removal only with a G:A-containing substrate. Both mMYH variants exhibit a significantly decreased affinity for duplexes containing noncleavable 2'-deoxyadenosine analogues. In addition, the human apurinic/apyrimidinic endonuclease (Ape1) stimulated product formation by wild-type and G365D mMYH with an OG:A substrate under conditions of multiple-turnover ([E]<[S]). In contrast, the presence of Ape1 nearly completely inhibited adenine removal by Y150C mMYH from the OG:A mismatch substrate. The more deleterious effect of Ape1 on the glycosylase activity of Y150C relative to G365D mMYH correlated with the more compromised binding affinity of Y150C to substrate analogue duplexes. These results suggest that the equivalent hMYH variants may be significantly compromised in substrate targeting in vivo due to a decrease in binding to substrate DNA; moreover, competition with other DNA binding proteins may further reduce the effective adenine glycosylase activity in vivo.     

Physical and functional interactions between MutY glycosylase homologue (MYH) and checkpoint proteins Rad9-Rad1-Hus1

The MYH (MutY glycosylase homologue) increases replication fidelity by removing adenines or 2-hydroxyadenine misincorporated opposite GO (7,8-dihydro-8-oxo-guanine). The 9-1-1 complex (Rad9, Rad1 and Hus1 heterotrimer complex) has been suggested as a DNA damage sensor. Here, we report that hMYH (human MYH) interacts with hHus1 (human Hus1) and hRad1 (human Rad1), but not with hRad9. In addition, interactions between MYH and the 9-1-1 complex, from both the fission yeast Schizosaccharomyces pombe and human cells, are partially interchangeable. The major Hus1-binding site is localized to residues 295-350 of hMYH and to residues 245-293 of SpMYH (S. pombe MYH). Val315 of hMYH and Ile261 of SpMYH play important roles for their interactions with Hus1. hHus1 protein and the 9-1-1 complex of S. pombe can enhance the glycosylase activity of SpMYH. Moreover, the interaction of hMYH-hHus1 is enhanced following ionizing radiation. A significant fraction of the hMYH nuclear foci co-localizes with hRad9 foci in H2O2-treated cells. These results reveal that the 9-1-1 complex plays a direct role in base excision repair.     

Increased risk of lung cancer associated with a functionally impaired polymorphic variant of the human DNA glycosylase NEIL2

Human NEIL2, one of five oxidized base-specific DNA glycosylases, is unique in preferentially repairing oxidative damage in transcribed genes. Here we show that depletion of NEIL2 causes a 6-7-fold increase in spontaneous mutation frequency in the HPRT gene of the V79 Chinese hamster lung cell line. This prompted us to screen for NEIL2 variants in lung cancer patients' genomic DNA. We identified several polymorphic variants, among which R103Q and R257L were frequently observed in lung cancer patients. We then characterized these variants biochemically, and observed a modest decrease in DNA glycosylase activity relative to the wild type (WT) only with the R257L mutant protein. However, in reconstituted repair assays containing WT NEIL2 or its R257L and R103Q variants together with other DNA base excision repair (BER) proteins (PNKP, Polβ, Lig IIIα and XRCC1) or using NEIL2-FLAG immunocomplexes, an ~5-fold decrease in repair was observed with the R257L variant compared to WT or R103Q NEIL2, apparently due to the R257L mutant's lower affinity for other repair proteins, particularly Polβ. Notably, increased endogenous DNA damage was observed in NEIL2 variant (R257L)-expressing cells relative to WT cells. Taken together, our results suggest that the decreased DNA repair capacity of the R257L variant can induce mutations that lead to lung cancer development.     

Abnormality in Wnt Signaling is Causatively Associated with Oxidative Stress-Induced Intestinal Tumorigenesis in MUTYH-Null Mice

MUTYH is a DNA glycosylase that excises adenine paired with 8-oxoguanine to prevent mutagenesis in mammals. Biallelic germline mutations of MUTYH have been found in patients predisposed to a recessive form of familial adenomatous polyposis (MAP: MUTYH-associated polyposis). We previously reported that Mutyh-deficient mice showed a high susceptibility to spontaneous and oxidative stress-induced intestinal adenoma/carcinoma. Here, we performed mutation analysis of the tumor-associated genes including Apc, Ctnnb1, Kras and Trp53 in the intestinal tumors of Mutyh-deficient mice. In the 62 tumors, we identified 25 mutations in Apc of 18 tumors and 36 mutations in Ctnnb1 of 36 tumors. Altogether, 54 out of the 62 tumors (87.1%) had a mutation in either Apc or Ctnnb1; no tumor displayed mutations simultaneously in the both genes. Similar to MAP, 60 out of 61 mutations (98.3%) were identified as G:C to T:A transversions of which 85% occurred at either AGAA or TGAA sequences. Immunohistochemical analyses revealed the accumulation of β-catenin in the nuclei of tumors. No mutation was found in either Kras or Trp53 in the tumors. These results indicate that the uncontrolled activation of Wnt signaling pathway is causatively associated with oxidative stress-induced intestinal tumorigenesis in the Mutyh-deficient mice.     

A single engineered point mutation in the adenine glycosylase MutY confers bifunctional glycosylase/AP lyase activity

The E. coli adenine glycosylase MutY is a member of the base excision repair (BER) superfamily of DNA repair enzymes. MutY plays an important role in preventing mutations caused by 7, 8-dihydro-8-oxo-2'-deoxyguanosine (OG) by removing adenine from OG:A base pairs. Some enzymes of the BER superfamily catalyze a strand scission even concomitant with base removal. These bifunctional glycosylase/AP lyases bear a conserved lysine group in the active site region, which is believed to be the species performing the initial nucleophilic attack at C1' in the catalysis of base removal. Monofunctional glycosylases such as MutY are thought to perform this C1' nucleophilic displacement by a base-activated water molecule, and, indeed, the conservation of amine functionality positioning has not been observed in protein sequence alignments. Bifunctional glycosylase/AP lyase activity was successfully engineered into MutY by replacing serine 120 with lysine. MutY S120K is capable of catalyzing DNA strand scission at a rate equivalent to that of adenine excision for both G:A and OG:A mispair substrates. The extent of DNA backbone cleavage is independent of treating reaction aliquots with 0.1 M NaOH. Importantly, the replacement of the serine with lysine results in a catalytic rate that is compromised by at least 20-fold. The reduced efficiency in the glycosylase activity is also reflected in a reduced ability of S120K MutY to prevent DNA mutations in vivo. These results illustrate that the mechanisms of action of the two classes of these enzymes are quite similar, such that a single amino acid change is sufficient, in the case of MutY, to convert a monofunctional glycosylase to a bifunctional glycosylase/AP lyase.     

A residue in MutY important for catalysis identified by photocross-linking and mass spectrometry

MutY is an adenine glycosylase in the base excision repair (BER) superfamily that is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA. MutY contains a [4Fe-4S]2+ cluster that is part of a novel DNA binding motif, referred to as the iron-sulfur cluster loop (FCL) motif. This motif is found in a subset of members of the BER glycosylase superfamily, defining the endonuclease III-like subfamily. Site-specific cross-linking was successfully employed to investigate the DNA-protein interface of MutY. The photoreactive nucleotide 4-thiothymidine (4ST) incorporated adjacent to the OG:A mismatch formed a specific cross-link between the substrate DNA and MutY. The amino acid participating in the cross-linking reaction was characterized by positive ion electrospray ionization (ESI) tandem mass spectrometry. This analysis revealed Arg 143 as the site of modification in MutY. Arg 143 and nearby Arg 147 are conserved throughout the endo III-like subfamily. Replacement of Arg 143 and Arg 147 with alanine by site-directed mutagenesis reduces adenine glycosylase activity of MutY toward OG:A and G:A mispairs. In addition, the R143A and R147A enzymes exhibit a reduced affinity for duplexes containing the substrate analogue 2'-deoxy-2'-fluoroadenosine opposite OG and G. Modeling of MutY bound to DNA using an endonuclease III-DNA complex structure shows that these two conserved arginines are located within close proximity to the DNA backbone. The insight from mass spectrometry experiments combined with functional mutagenesis results indicate that these two amino acids in the [4Fe-4S]2+ cluster-containing subfamily play an important role in recognition of the damaged DNA substrate.     

Structure and stereochemistry of the base excision repair glycosylase MutY reveal a mechanism similar to retaining glycosidases

MutY adenine glycosylases prevent DNA mutations by excising adenine from promutagenic 8-oxo-7,8-dihydroguanine (OG):A mismatches. Here, we describe structural features of the MutY active site bound to an azaribose transition state analog which indicate a catalytic role for Tyr126 and approach of the water nucleophile on the same side as the departing adenine base. The idea that Tyr126 participates in catalysis, recently predicted by modeling calculations, is strongly supported by mutagenesis and by seeing close contact between the hydroxyl group of this residue and the azaribose moiety of the transition state analog. NMR analysis of MutY methanolysis products corroborates a mechanism for adenine removal with retention of stereochemistry. Based on these results, we propose a revised mechanism for MutY that involves two nucleophilic displacement steps akin to the mechanisms accepted for ‘retaining’ O-glycosidases. This new-for-MutY yet familiar mechanism may also be operative in related base excision repair glycosylases and provides a critical framework for analysis of human MutY (MUTYH) variants associated with inherited colorectal cancer.     

Coordination of MYH DNA glycosylase and APE1 endonuclease activities via physical interactions

MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G^o) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugar–phosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5′ to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9–Rad1–Hus1 (9–1–1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein–DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates.     

Insight into the functional consequences of inherited variants of the hMYH adenine glycosylase associated with colorectal cancer: complementation assays with hMYH variants and pre-steady-state kinetics of the corresponding mutated E.coli enzymes

The oxidized guanine lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) is highly mutagenic, resulting in G:C to T:A transversion mutations in the absence of repair. The Escherichia coli adenine glycosylase MutY and its human homolog (hMYH) play an important role in the prevention of mutations associated with OG by removing misincorporated adenine residues from OG:A mismatches. Previously, biallelic mutations of hMYH have been identified in a British family (Family N) with symptoms characteristic of familial adenomatous polyposis (FAP), which is typically associated with mutations in the adenomatous polyposis coli (APC) gene. Afflicted members of this family were compound heterozygotes for two mutations in hMYH, Y165C and G382D. These positions are highly conserved in MutY across phylogeny. The current work reveals a reduced ability of the hMYH variants compared to wild-type (WT) hMYH to complement the activity of E.coli MutY in mutY((-)) E.coli. In vitro analysis of the corresponding mutations in E.coli MutY revealed a reduction in the adenine glycosylase activity of the enzymes. In addition, evaluation of substrate affinity using a substrate analog, 2'-deoxy-2'-fluoroadenosine (FA) revealed that both mutations severely diminish the ability to recognize FA, and discriminate between OG and G. Importantly, adenine removal with both the mutant and WT E.coli enzymes was observed to be less efficient from a mismatch in the sequence context observed to be predominantly mutated in tumors of Family N. Interestingly, the magnitude of the reduced activity of the E.coli mutant enzymes relative to the WT enzyme was magnified in the "hotspot" sequence context. If the corresponding mutations in hMYH cause similar sensitivity to sequence context, this effect may contribute to the specific targeting of the APC gene. The lack of complementation of the hMYH variants for MutY, and the reduced activity of the Y82C and G253D E.coli enzymes, provide additional circumstantial evidence that the somatic mutations in APC, and the occurrence of FAP in Family N, are due to a reduced ability of the Y165C and G382D hMYH enzymes to recognize and repair OG:A mismatches.     

The value of MUTYH testing in patients with early onset microsatellite stable colorectal cancer referred for hereditary nonpolyposis colon cancer syndrome testing

MUTYH adenomatous polyposis (MAP) can mimic both the familial adenomatous polyposis (FAP) and hereditary nonpolyposis colon cancer (HNPCC) phenotypes. As a result of MAP's phenotypic overlap with FAP, some DNA diagnostic laboratories perform MUTYH testing in conjunction with APC testing in patients with suspected FAP or attenuated FAP (AFAP). In addition to testing FAP/AFAP samples for MUTYH mutations, we were interested whether there would also be value in testing samples referred for HNPCC testing. To determine this, we tested a consecutive series of 229 samples referred for HNPCC testing for the two most common MUTYH mutations in the Caucasian population. To enrich our study population with MAP cases, we only included samples from patients with early onset colorectal cancer (CRC diagnosed <50 years old) in whom HNPCC had been excluded by microsatellite instability testing (microsatellite stable or low microsatellite instability). Four biallelic (2%) and six monoallelic (3%) MUTYH mutation carriers were identified. No clinical factors predicted MUTYH mutation status. Specifically, a family history of vertical transmission of CRC or having few polyps (<15) did not rule out the possibility of biallelic MUTYH mutations. Thus, MUTYH mutation testing may be a reasonable cascade test in early onset CRC found to have proficient DNA mismatch repair, regardless of pattern of family history or number of polyps.     

A functional analysis of the DNA glycosylase activity of mouse MUTYH protein excising 2-hydroxyadenine opposite guanine in DNA

2-Hydroxy-2-deoxyadenosine triphosphate (2-OH-dATP), generated by the oxidation of dATP, can be misincorporated by DNA polymerases opposite guanine in template DNA during DNA replication, thus causing spontaneous mutagenesis. We demonstrated that mouse MUTYH (mMUTYH) has a DNA glycosylase activity excising not only adenine opposite 8-oxoguanine (8-oxoG) but also 2-hydroxyadenine (2-OH-A) opposite guanine, using purified recombinant thioredoxin-mMUTYH fusion protein. mMUTYH formed a stable complex with duplex oligonucleotides containing an adenine:8-oxoG pair, but the binding of mMUTYH to oligonucleotides containing a 2-OH-A:guanine pair was barely detectable, thus suggesting that mMUTYH recognizes and interacts with these two substrates in a different manner which may reflect the difference in the base excision repair process for each substrate. Mutant mMUTYH with G365D amino acid substitution, corresponding to a G382D germline mutation of human MUTYH found in familial adenomatous polyposis patients, almost completely retained its DNA glycosylase activity excising adenine opposite 8-oxoG; however, it possessed 1.5% of the wild-type activity excising 2-OH-A opposite guanine. Our results imply that the reduced repair capacity of the mutant hMUTYH(G382D), which inefficiently excises 2-OH-A opposite guanine, results in an increased occurrence of somatic G:C to T:A transversion mutations in the APC gene as well as tumorigenesis in the colon.