SCML2 inhibits PrimPol-mediated DNA replication repriming and regulates replication gap-associated cancer cell vulnerability.

PrimPol-mediated fork repriming generates single-stranded DNA (ssDNA) gaps during DNA damage tolerance (DDT), which, if not properly repaired, can threaten genomic stability. PrimPol is a highly error-prone polymerase. Thus, PrimPol activity should be tightly regulated to fit into DDT and remain low during unstressed DNA replication. Human sex comb on midleg like-2 (SCML2), a Polycomb protein containing MBT repeats, has been identified as a component of the noncanonical PRC1 complex and a regulator of p53 and CHK1 stability. Here, we show that SCML2A is a suppressor of PrimPol. SCML2A directly binds two domains of PrimPol: Its RBR domain binds the C terminus of PrimPol, and its SAM domain binds the amino-terminal AEP domain, thereby inhibiting ssDNA-binding capacity of the AEP domain. SCML2 depletion leads to increased nascent DNA synthesis and accumulation of PrimPol-mediated ssDNA gaps under replication stress. SCML2 depletion also enhances PrimPol engagement during unstressed DNA replication, leading to subsequent accumulation of ssDNA gaps, which are filled by REV1/POLζ-mediated translesion synthesis (TLS) and RAD51-mediated template switching (TS). SCML2 depletion confers cellular hypersensitivity to RAD51 or REV1/POLζ inhibition, which is associated with increased accumulation of ssDNA gaps. Furthermore, CDK2 phosphorylates SCML2A at Ser267, which suppresses RBR-mediated PrimPol interaction. Cyclin E1 overexpression leads to CDK2-mediated SCML2A Ser267 hyperphosphorylation, which releases the inhibitory effect of SCML2A on PrimPol and subsequently induces ssDNA gap accumulation. Collectively, we identify SCML2A as a negative regulator of PrimPol.