Abstract
Eukaryotic Primase-Polymerase (PrimPol) is an enzyme that maintains efficient DNA duplication by repriming replication restart downstream of replicase stalling lesions and structures. To elucidate the cellular requirements for PrimPol in human cells, we generated PrimPol-deleted cell lines and show that it plays key roles in maintaining active replication in both the nucleus and mitochondrion, even in the absence of exogenous damage. Human cells lacking PrimPol exhibit delayed recovery after UV-C damage and increased mutation frequency, micronuclei and sister chromatin exchanges but are not sensitive to genotoxins. PrimPol is also required during mitochondrial replication, with PrimPol-deficient cells having increased mtDNA copy number but displaying a significant decrease in replication. Deletion of PrimPol in XPV cells, lacking functional polymerase Eta, causes an increase in DNA damage sensitivity and pronounced fork stalling after UV-C treatment. We show that, unlike canonical TLS polymerases, PrimPol is important for allowing active replication to proceed, even in the absence of exogenous damage, thus preventing the accumulation of excessive fork stalling and genetic mutations. Together, these findings highlight the importance of PrimPol for maintaining efficient DNA replication in unperturbed cells and its complementary roles, with Pol Eta, in damage tolerance in human cells.
Highlights
To successfully maintain genome integrity, cells must accurately and efficiently replicate their DNA to pass on accurate copies to daughter cells
Generation of a human PrimPol Knockout cell line Previous human PrimPol depletion studies, using siRNA-knockdown, suggested that it has a role in DNA damage tolerance within the nucleus, as well being involved in the maintenance of mtDNA [10,11,12,15,16]
We observed a consistent decrease in cell growth rate after reduction of PrimPol by siRNA, compared to cells treated with a scrambled siRNA control, suggesting an important role in maintaining cell proliferation (Figure 1A and Supplementary Figure S1A)
Summary
To successfully maintain genome integrity, cells must accurately and efficiently replicate their DNA to pass on accurate copies to daughter cells. Loss of human PrimPol does not overtly affect cell viability after DNA damage, the additional loss of Pol Eta (h) results in increased damage sensitivity, delayed recovery and enhanced fork stalling.
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