title: DNA damage recognition and repair by the murine MutY homologue.
abstract: E. coli MutY excises adenine from duplex DNA when it is mispaired with the mutagenic oxidative lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG). While E. coli MutY has been extensively studied, a detailed kinetic analysis of a mammalian MutY homologue has been inhibited by poor overexpression in bacterial hosts. This current work is the first detailed study of substrate recognition and repair of mismatched DNA by a mammalian adenine glycosylase, the murine MutY homologue (mMYH). Similar to E. coli MutY, the processing of OG:A substrates by mMYH is biphasic, indicating that product release is rate-limiting. Surprisingly, the intrinsic rates of adenine removal from both OG:A and G:A substrates by mMYH are diminished ( approximately 10-fold) compared to E. coli MutY. However, similar to E. coli MutY, the rate of adenine removal is approximately nine-fold faster with an OG:A- than a G:A-containing substrate. Interestingly, the rate of removal of 2-hydroxyadenine mispaired with OG or G in duplex DNA by mMYH was similar to the rate of adenine removal from the analogous context. In contrast, 2-hydroxyadenine removal by E. coli MutY was significantly reduced compared to adenine removal opposite both OG and G. Furthermore, dissociation constant measurements with duplexes containing noncleavable 2'-deoxyadenosine analogues indicate that mMYH is less sensitive to the structure of the base mispaired with OG or G than MutY. Though in many respects the catalytic behavior of mMYH is similar to E. coli MutY, the subtle differences may translate into differences in their in vivo functions.
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Pope
authors.forename: Sheila S
authors.initials: SS
authors.lastname: David
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D000225:Adenine / Q000378:metabolism; D000818:Animals; D020137:Base Pair Mismatch / Q000235:genetics*; D004249:DNA Damage; D018983:DNA Footprinting; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli; D007700:Kinetics; D051379:Mice / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D009841:Oligonucleotides; D000080242:8-Hydroxy-2'-Deoxyguanosine; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase; D003849:Deoxyguanosine; D000225:Adenine