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

Among human four Y-family DNA polymerases, hPol ι is exceptionally error-prone in DNA synthesis. 6 mA 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 6 mA and Hyp, the intermediate of 6 mA, inhibited the replication of DNA by hPol ι. 6 mA lost priority in extension beyond 6 mA: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, 6 mA primarily reduced the burst incorporation rate (k_pol) and slightly increased the dissociation constant (K_d,dTTP). However, Hyp mainly increased the K_d,dCTP yet did not affect the k_pol, both reducing the burst incorporation efficiency (k_pol/K_d,dCTP). 6 mA together with Hyp weakened the binding affinity of hPol ι to DNA in binary or ternary complex. The misincorporation opposite 6 mA or Hyp further weakened this binding affinity. The methyl group in 6 mA 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 6 mA and its intermediate Hyp affect replication of DNA by human DNA polymerase ι.