Epigenetically modified N6-methyladenine inhibits DNA replication by human DNA polymerase iota

ABSTRACT Among human four Y-family DNA polymerases, hPol ι is exceptionally error-prone in DNA synthesis. 6mA plays significant roles in epigenetic regulation of numerous biological processes. Nonetheless, its effects on DNA replication by hPol ι is still unclear. In this work, we found that 6mA and Hyp, the intermediate of 6mA, inhibited the replication of DNA by hPol ι. 6mA lost priority in extension beyond 6mA:T pair, partially reducing dTTP incorporation efficiency and inhibiting next-base extension. Hyp was prone to dCTP incorporation and extension beyond Hyp:C instead of Hyp:T pair. Statistically, 6mA primarily reduced the burst incorporation rate (kpol) and slightly increased the dissociation constant (Kd,dTTP). However, Hyp mainly increased the Kd,dCTP yet did not affect the kpol, both reducing the burst incorporation efficiency (kpol/Kd,dCTP). 6mA together with Hyp weakened the binding affinity of hPol ι to DNA in binary or ternary complex. The misincorporation opposite 6mA or Hyp further weakened this binding affinity. The methyl group in 6mA doesn’t almost affect the H-bond formation with dTTP, therefore mildly inhibiting dTTP incorporation. As an analogue of G, Hyp can form only two H-bonds with dCTP, thus reducing dCTP incorporation. This work provides a new insight in how the epigenetically modified 6mA and its intermediate Hyp affect replication of DNA by human DNA polymerase ι.