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

<p>PrimPol is a DNA damage tolerance enzyme possessing both translesion synthesis (TLS) and primase activities. To uncover its potential role in TLS-mediated IgV_λ hypermutation and define its interplay with other TLS polymerases, <i>PrimPol</i>^−/− and <i>PrimPol</i>^−/−<i>/Pol</i>η^−/−<i>/Pol</i>ζ ^−/− gene knockouts were generated in avian cells. Loss of PrimPol had no significant impact on the rate of hypermutation or the mutation spectrum of IgV_λ. However, <i>PrimPol</i>^−/− cells were sensitive to methylmethane sulfonate, suggesting that it may bypass abasic sites at the IgV_λ segment by repriming DNA synthesis downstream of these sites. <i>PrimPol</i>^−/− 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 <i>PrimPol</i>^−/− cells complemented with polymerase or primase-deficient PrimPol. Polymerase-defective, but not primase-deficient, PrimPol suppresses the hypersensitivity of <i>PrimPol</i>^−/− 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 <i>PrimPol</i>^−/− cells. Moreover, we found that PrimPol and Polη-Polζ redundantly prevented cell death and facilitated unperturbed cell cycle progression. <i>PrimPol</i>^−/− 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 <i>in vitro</i>. Together, these results indicate that PrimPol's repriming activity plays a central role in reinitiating replication downstream from CTNAs and other specific DNA lesions.