Mechanisms by which herpes simplex virus DNA polymerase limits translesion synthesis through abasic sites

Abstract

Results suggest a high probability that abasic (AP) sites occur at least once per herpes simplex virus type 1 (HSV-1) genome. The parameters that control the ability of HSV-1 DNA polymerase (pol) to engage in AP translesion synthesis (TLS) were examined because AP lesions could influence the completion and fidelity of viral DNA synthesis. Pre-steady-state kinetic experiments demonstrated that wild-type (WT) and exonuclease-deficient (exo -) pol could incorporate opposite an AP lesion, but full TLS required absence of exo function. Virtually all of the WT pol was bound at the exo site to AP-containing P/Ts at equilibrium, and the pre-steady-state rate of excision by WT pol was higher on AP-containing than on matched DNA. However, several factors influencing polymerization work synergistically with exo activity to prevent HSV-1 pol from engaging in TLS. Although the pre-steady-state catalytic rate constant for insertion of dATP opposite a T or AP site was similar, ground-state binding affinity of dATP for insertion opposite an AP site was reduced 3−9-fold. Single-turnover running-start experiments demonstrated a reduced proportion of P/Ts extended to the AP site compared to the preceding site during processive synthesis by WT or exo -pol. Only the exo -pol engaged in TLS, though inefficiently and without burst kinetics, suggesting a much slower rate-limiting step for extension beyond the AP site.

DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity

“…The bypass of abasic sites by replicative DNA polymerases is very low, depending on the sequence context and counteracted by the proofreading activity (12,13,40,41). Fig.…”

“…Unrepaired abasic sites are highly blocking lesions for replicative DNA polymerases (10), although mutant polymerases with impaired proofreading activity or with mutations in the polymerization active site residues that affect the incoming nucleotide selection have been shown to have enhanced AP site bypass capacity (11)(12)(13)(14)(15).…”

The replicative DNA polymerase of herpes simplex virus 1 exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities

“…These may arise through spontaneous base loss and as a result of DNA glycosylase action, presumably that of the virus-encoded UL2. Because AP sites are noninstructional and cause UL30 to stall (10), their occurrence in the viral genome necessitates that they are repaired by BER. Here, we have shown that UL30, a replicative B family Pol and a Pol from the herpesviridae family, possesses both AP and 5Ј dRP lyase activities that are integral to BER.…”

“…In the case of HSV-1, a recent study showed that DNA isolated from HSV-1-infected cultured fibroblasts contains a steady state of 2.8-5.9 AP sites per viral genome equivalent (10). Because AP sites are noninstructional lesions, they may either induce mutagenesis or cause replisome stalling.…”

“…Because AP sites are noninstructional lesions, they may either induce mutagenesis or cause replisome stalling. Indeed, the HSV-1 Pol (UL30) cannot replicate beyond a model AP site (tetrahydrofuran residue) (10), indicating the necessity to repair this type of lesion. In mammalian singlenucleotide base excision repair (BER) initiated by monofunctional DNA glycosylases, AP sites are incised hydrolytically at the 5Ј side by AP endonuclease (APE) followed by one nucleotide addition by Pol ␤ to generate a 5Јdeoxyribose phosphate (dRP) flap (11).…”