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.
"Several variants have been identified in MAP patients (13), and the mechanism underlying a defective enzymatic function has been extensively investigated by monitoring the DNA–glycosylase activity on synthetic DNA substrates. Recombinant bacterial (11), human (14–19), murine (20–22) MUTY/MUTYH proteins, as well as lysates derived from lymphoblastoid cell lines from MAP patients, (23,24) were used. The most common variants have been found totally or partially devoid of DNA–glycosylase activity, and the adenine removal capability is generally taken as a functional biomarker. "
[Show abstract][Hide abstract] ABSTRACT: The MUTYH DNA-glycosylase is indirectly engaged in the repair of the miscoding 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxodG) lesion by removing adenine erroneously incorporated opposite the oxidized purine. Inherited biallelic mutations in the MUTYH gene are responsible for a recessive syndrome, the MUTYH-associated polyposis (MAP), which confers an increased risk of colorectal cancer. In this study, we functionally characterized the Q338H variant using recombinant proteins, as well as cell-based assays. This is a common variant among human colorectal cancer genes, which is generally considered, unrelated to the MAP phenotype but recently indicated as a low-penetrance allele. We demonstrate that the Q338H variant retains a wild-type DNA-glycosylase activity in vitro, but it shows a reduced ability to interact with the replication sensor RAD9:RAD1:HUS1 (9-1-1) complex. In comparison with Mutyh-/- mouse embryo fibroblasts expressing a wild-type MUTYH cDNA, the expression of Q338H variant was associated with increased levels of DNA 8-oxodG, hypersensitivity to oxidant and accumulation of the population in the S phase of the cell cycle. Thus, an inefficient interaction of MUTYH with the 9-1-1 complex leads to a repair-defective phenotype, indicating that a proper communication between MUTYH enzymatic function and the S phase checkpoint is needed for effective repair of oxidative damage.
Nucleic Acids Research 03/2013; 41(7). DOI:10.1093/nar/gkt130 · 9.11 Impact Factor
"Owing to the capacity of MUTYH to recognize and excise adenine from DNA duplexes containing either 8-oxoG:A or G:2-OH-A mismatches, in vitro tests with purified proteins have been performed . By using different oligonucleotides containing specific mismatches as substrate, the enzymatic activities of bacterial (Al-Tassan et al., 2002; Chmiel et al., 2003; Livingston et al., 2005), mouse (Hirano et al., 2003; Pope et al., 2005; Ushijima et al., 2005; Yanaru-Fujisawa et al., 2008), and human (Bai et al., 2005, 2007; Ali et al., 2008; Kundu et al., 2009; D'Agostino et al., 2010) purified proteins have been investigated. The glycosylase activity has been evaluated by measuring the amount of cleavage fragments derived from DNA duplexes, while the DNA-binding capacity has mainly been investigated through the formation of DNA-protein complexes in electrophoresis mobility shift assay (EMSA). "
[Show abstract][Hide abstract] ABSTRACT: In 2002, Al-Tassan and co-workers described for the first time a recessive form of inherited polyposis associated with germline mutations of MUTYH, a gene encoding a base excision repair (BER) protein that counteracts the DNA damage induced by the oxidative stress. MUTYH-associated polyposis (MAP) is now a well-defined cancer susceptibility syndrome, showing peculiar molecular features that characterize disease progression. However, some aspects of MAP, including diagnostic criteria, genotype-phenotype correlations, pathogenicity of variants, as well as relationships between BER and other DNA repair pathways, are still poorly understood. A deeper knowledge of the MUTYH expression pattern is likely to refine our understanding of the protein role and, finally, to improve guidances for identifying and handling MAP patients.
Frontiers in Oncology 08/2012; 2:83. DOI:10.3389/fonc.2012.00083
"Our laboratory has previously exploited the use of non-cleavable analogues to reveal fundamental features concerning recognition of bacterial and mammalian MutY enzymes      . In previous studies, the FA α nucleotide, where α designates the ribo sugar conformation, was used in place of A while in the present study, the FA β analogue, where β designates the arabino sugar conformation, was used. "
[Show abstract][Hide abstract] ABSTRACT: 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.
DNA repair 10/2010; 9(10):1026-37. DOI:10.1016/j.dnarep.2010.07.002 · 3.11 Impact Factor
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