Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides

Abstract

PrimPol is a DNA damage tolerance enzyme possessing both translesion synthesis (TLS) and primase activities. To uncover its potential role in TLS-mediated IgVλ hypermutation and define its interplay with other TLS polymerases, PrimPol−/− and PrimPol−/−/Polη−/−/Polζ −/− gene knockouts were generated in avian cells. Loss of PrimPol had no significant impact on the rate of hypermutation or the mutation spectrum of IgVλ. However, PrimPol−/− cells were sensitive to methylmethane sulfonate, suggesting that it may bypass abasic sites at the IgVλ segment by repriming DNA synthesis downstream of these sites. PrimPol−/− cells were also sensitive to cisplatin and hydroxyurea, indicating that it assists in maintaining / restarting replication at a variety of lesions. To accurately measure the relative contribution of the TLS and primase activities, we examined DNA damage sensitivity in PrimPol−/− cells complemented with polymerase or primase-deficient PrimPol. Polymerase-defective, but not primase-deficient, PrimPol suppresses the hypersensitivity of PrimPol−/− cells. This indicates that its primase, rather than TLS activity, is pivotal for DNA damage tolerance. Loss of TLS polymerases, Polη and Polζ has an additive effect on the sensitivity of PrimPol−/− cells. Moreover, we found that PrimPol and Polη-Polζ redundantly prevented cell death and facilitated unperturbed cell cycle progression. PrimPol−/− cells also exhibited increased sensitivity to a wide variety of chain-terminating nucleoside analogs (CTNAs). PrimPol could perform close-coupled repriming downstream of CTNAs and oxidative damage in vitro. Together, these results indicate that PrimPol's repriming activity plays a central role in reinitiating replication downstream from CTNAs and other specific DNA lesions.

Molecular basis for PrimPol recruitment to replication forks by RPA

“…However, during primer synthesis the ZnF domain can access ssDNA both upstream and downstream of the AEP domain. Recent studies highlighting the importance of PrimPol's primase activity in vivo 9101112, coupled with the recruitment of the enzyme via RPA70N shown here, argue that PrimPol more likely binds downstream of RPA, with the ZnF bound to ssDNA upstream of the AEP domain, during primer synthesis (Fig. 7b).…”

Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle

“…Hence, PrimPol may serve to re-prime leading strand templates downstream of UV-induced lesions. Such activity had been demonstrated in vitro with various obstructions that block DNA synthesis and, indeed, primers synthesized by PrimPol can be efficiently extended by pol ε 141, 144, 145 . Clever genetic assays validated this model in vivo .…”

“…Similar behavior was also observed for PrimPol after UV treatment and PrimPol foci colocalized with UV-induced lesions and PCNA 73, 74 . Survival assays revealed that depletion or knockout of PrimPol sensitizes cells to UV treatment and such behavior was non-epistatic with both pol η and pol ζ 74, 141, 144 . However, re-localization of PrimPol was not observed after DNA strand breaks were generated by ionizing radiation (IR) and PrimPol knockout cells are not sensitive to IR 74 .…”

“…PrimPol knockout cells are hypersensitive to killing by CTNA treatment and suppression of this phenotype by complementation absolutely required the primase activity of PrimPol 144 . The same behavior was also observed with UV treatment, suggesting that re-priming of damaged DNA templates by PrimPol is critical for the cellular tolerance of UV-induced lesions 74, 141, 144 .…”

“…PrimPol knockout cells are hypersensitive to killing by CTNA treatment and suppression of this phenotype by complementation absolutely required the primase activity of PrimPol 144 . The same behavior was also observed with UV treatment, suggesting that re-priming of damaged DNA templates by PrimPol is critical for the cellular tolerance of UV-induced lesions 74, 141, 144 . Indeed, loss of PrimPol markedly increased the delay or blockage of replication fork progression in UV-irradiated cells and reversion of this phenotype was only achieved by complementation with PrimPol containing active primase activity 73, 74, 142, 144 .…”