Motif WFYY of human PrimPol is crucial to stabilize the incoming 3′-nucleotide during replication fork restart

PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp87 and Tyr90 residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3′-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart.BFU2015-65880-P (MINECO) and PGC2018-093576-B.C21 (MCI/AEI/FEDER,UE) to L.B., BFU2016-80402-R (MINECO/FEDER, UE) and PID2019-106707RB-100 (AEI/10.13039/501100011033) to J.M., Kempe JCK 1831 to G.S, Knut och Alice Wallenbergs Foundation KAW 2019.0307, VR-2018-02781, to S.W., and by institutional grants from Fundación Ramón Areces and Banco de Santande