Effect of 8-oxoguanine and abasic site DNA lesions on in vitro elongation by human DNA polymerase ϵ in the presence of replication protein A and proliferating-cell nuclear antigen

DNA pol (polymerase) is thought to be the leading strand replicase in eukaryotes. In the present paper, we show that human DNA pol can efficiently bypass an 8-oxo-G (7,8-dihydro-8-oxoguanine) lesion on the template strand by inserting either dCMP or dAMP opposite to it, but it cannot bypass an abasic site. During replication, DNA pols associate with accessory proteins that may alter their bypass ability. We investigated the role of the human DNA sliding clamp PCNA (proliferating-cell nuclear antigen) and of the human single-stranded DNA-binding protein RPA (replication protein A) in the modulation of the DNA synthesis and translesion capacity of DNA pol . RPA inhibited the elongation by human DNA pol on templates annealed to short primers. PCNA did not influence the elongation by DNA pol and had no effect on inhibition of elongation caused by RPA. RPA inhibition was considerably reduced when the length of the primers was increased. On templates bearing the 8-oxo-G lesion, this inhibitory effect was more pronounced on DNA replication beyond the lesion, suggesting that RPA may prevent extension by DNA pol after incorporation opposite an 8-oxo-G. Neither PCNA nor RPA had any effect on the inability of DNA pol to replicate past the AP site, independent of the primer length.