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.
Pope
Mary Ann
MA
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112, USA.
Chmiel
Nikolas H
NH
David
Sheila S
SS
D000818:Animals; D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000235:genetics*; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D006801:Humans; D051379:Mice; D013379:Substrate Specificity
D017931:DNA Primers; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase