The Kinetic Studying Of Epigenetically Modified N~6-methyladenine Inhibits DNA Replication Catalyzed By Human DNA Polymerase ?

N6-methyladenine(6m A)was newly reported as a novel epigenetic marker,which plays significant roles in regulation of various biological processes in eukaryotes.However,the effect of 6m A on human DNA replication remain elsive.In this work,we used Y-family human DNA polymerase ? as a model to investigate the mechanism of bypass of 6m A by h Pol ? through both biochemical and biophysical approaches.We found 6m A and its intermediate hypoxanthine(I)on template partially inhibited DNA replication by h Pol ?.6m A reduced d TTP incorporation efficiency by 13-fold and next-base extension efficiency by 8.5-fold.I on template preferred to d CTP incorporation.h Pol ? lost its priority in extension beyond 6m A:T pair than 6m A:C mispair and preferred to extension beyond I:C rather than I:T pair.d TTP incorporation opposite 6m A and d CTP opposite I showed fast burst phases.However,6m A and I reduced the burst incorporation efficiency(kpol/Kd,d CTP)by 2.3-fold and 5.6-fold,respectively,compared with that of d TTP incorporation opposite A,due to the reduced incorporation rate(kpol)and increased dissociation constant(Kd,d NTP).Biophysical binding assays revealed that6 m A and I on template reduced the binding affinity of h Pol ? to DNA in both binary and ternary complexes compared with unmodified A.Incorrect base pair of d CTP with A or6 m A,or d TTP with I,further weakened the binding affinity of polymerase to DNA relative to that of correct base pair of d TTP with A or 6m A,or d CTP with I.In conclusion,we found 6m A can decrease the d NTP incorporation efficiency,reduce the binding affinity of d TTP to polymerase?DNA complex and weaken the binding affinity of polymerase to DNA in ternary complex,thereby decreasing the fraction of the productive enzyme?DNA complex and the average extension rate.Therefore,6m A in template inhibits DNA replication by human polymerase ?.This study also provides a new insight on the effects of epigenetically modified 6m A on human DNA replication.