PrimPol is required for the maintenance of efficient nuclear and mitochondrial DNA replication in human cells

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 rates, 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 rates. Deletion of PrimPol in XPV cells, lacking functional Pol 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.(200 words)

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

“…S7C). This is in line with previous findings that PrimPol-depleted human cells are not very sensitive to DNA damage induced by UV, suggesting that other pathways such as TLS or fork regression can compensate 12, 35 . It also suggests that the PrimPol-mediated pathway might predominantly impact on BPDE-induced mutagenesis and/or the level of genomic rearrangements, rather than cytotoxicity.…”

“…U2OS, A549 and MRC5 cells were obtained from ATCC. The SW480SN.3 52 , PrimPol -/- MRC5 35 , and U2OS cell lines carrying doxycycline (Dox)-inducible PrimPol shRNA 11 have been described before. Cells were confirmed to be free of Mycoplasma infection and grown in Dulbecco’s modified Eagle’s Medium (DMEM) with 10% foetal bovine serum in a humidified atmosphere containing 5% CO 2 .…”

Mitochondrial DNA: Epigenetics and environment

“…The Polγ holoenzyme is a heterotrimer consisting of two identical accessory subunits (Polγ-B) and one catalytic subunit (Polγ-A), where the accessory subunits enhance the processivity and catalytic function of Polγ-A (Gray and Wong, 1992;Johnson et al, 2000;Carrodeguas et al, 2002;reviewed in Kaguni, 2004). Until recently, Polγ was thought to be the sole polymerase within the mitochondrion that was responsible for both mtDNA replication and repair; however, at least four other DNA polymerases have been noted to play a role in mtDNA maintenance namely, DNA polymerase beta, PrimPol, polymerase theta, and polymerase zeta (Singh et al, 2015;Wisnovsky et al, 2016;Sykora et al, 2017;Bailey et al, 2019;reviewed in Bailey and Doherty, 2017;Kaufman and Van Houten, 2017;Krasich and Copeland, 2017). Until recently, Polγ was thought to be the sole polymerase within the mitochondrion that was responsible for both mtDNA replication and repair; however, at least four other DNA polymerases have been noted to play a role in mtDNA maintenance namely, DNA polymerase beta, PrimPol, polymerase theta, and polymerase zeta (Singh et al, 2015;Wisnovsky et al, 2016;Sykora et al, 2017;Bailey et al, 2019;reviewed in Bailey and Doherty, 2017;Kaufman and Van Houten, 2017;Krasich and Copeland, 2017).…”

Crystal structure and molecular dynamics of human POLDIP2, a multifaceted adaptor protein in metabolism and genome stability

“…2 PrimPol is an archaeo-eukaryotic primase involved in genome stability in both the nucleus and mitochondria. 9 POLDIP2 directly stimulates PrimPol DNA binding, synthesis and processivity during translesion bypass. 8 Furthermore, POLDIP2 also interacts with multiple mitochondrial components and influences mitochondrial morphology.…”

“…POLDIP2 stimulates Polδ by increasing its binding to PCNA, a homotrimeric ring‐shaped processivity factor for DNA synthesis, 7 potentially by bridging the molecules, with the POLDIP2 NTD required for full stimulation 2 . PrimPol is an archaeo‐eukaryotic primase involved in genome stability in both the nucleus and mitochondria 9 . POLDIP2 directly stimulates PrimPol DNA binding, synthesis and processivity during translesion bypass 8 .…”