DNA damage recognition and repair by the murine MutY homologue.
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.
Pope
Mary Ann
MA
Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
David
Sheila S
SS
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
D009841:Oligonucleotides; D000080242:8-Hydroxy-2'-Deoxyguanosine; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase; D003849:Deoxyguanosine; D000225:Adenine