| 2013 |
PrimPol is a second primase in human cells capable of initiating DNA chains with deoxynucleotides (unlike regular primases that use ribonucleotides), possesses DNA polymerase activity capable of bypassing oxidative lesions (abasic sites and 8-oxoguanine), and is present in both nuclear and mitochondrial DNA compartments. PrimPol activity is absent from mitochondria derived from PRIMPOL knockout mice, and PRIMPOL gene silencing impairs mitochondrial DNA replication. Synergy observed with replicative DNA polymerases Polγ and Polε supports a role in facilitating replication fork progression. |
In vitro primase/polymerase assays, subcellular fractionation, immunodetection, mitochondrial lysate activity assays, PRIMPOL knockout mice |
Molecular cell |
High |
24207056
|
| 2013 |
PrimPol uses its primase activity to mediate uninterrupted replication fork progression after UV irradiation and to reinitiate DNA synthesis after dNTP depletion, acting as a repriming enzyme downstream of lesions at stalled replication forks. |
DNA fiber analysis, UV irradiation assays, dNTP depletion experiments, siRNA knockdown |
Nature structural & molecular biology |
High |
24240614
|
| 2013 |
PrimPol (CCDC111) is involved in chromosomal DNA replication and is required for replication fork progression on UV-damaged DNA templates, mediating bypass of UV photoproducts. This bypass pathway is not epistatic with the Polη-dependent pathway, and PrimPol is also required for efficient replication fork progression during unperturbed S phase. |
Genetic knockdown/knockout (siRNA, DT40 cells), DNA fiber assays, epistasis analysis with Pol η, colony survival assays in XP-V cells |
Molecular cell |
High |
24267451
|
| 2013 |
hPrimpol1 possesses primase and DNA polymerase activities in vitro, directly interacts with RPA1, and is recruited to sites of DNA damage and stalled replication forks in an RPA1-dependent manner. Cells depleted of hPrimpol1 display increased spontaneous DNA damage and defects in restart of stalled replication forks. Both RPA1 binding and primase activity are required for cellular function. |
In vitro primase/polymerase assays, Co-IP (RPA1 interaction), immunofluorescence at damage sites, siRNA knockdown, complementation with activity mutants |
EMBO reports |
High |
24126761
|
| 2014 |
The zinc finger domain (ZnFD) of human PrimPol binds zinc ions and is essential for primase activity but dispensable for polymerase activity; it regulates processivity and fidelity of extension. The polymerase domain binds both ssDNA and dsDNA while the ZnFD binds only ssDNA. PrimPol's primase activity is required to restore wild-type replication fork rates in irradiated PrimPol−/− cells, while polymerase activity is sufficient for regular replisome progression in unperturbed cells. |
Domain deletion/mutagenesis, in vitro primase and polymerase assays, metal-binding assays, DNA binding assays, DNA fiber analysis in PrimPol−/− cells complemented with separation-of-function mutants |
Nucleic acids research |
High |
24682820
|
| 2014 |
PrimPol is not stimulated by PCNA and does not interact with PCNA in vivo. PrimPol interacts with both major single-strand binding proteins RPA and mtSSB in vivo. By NMR spectroscopy, PrimPol binds directly to the N-terminal domain of RPA70. SSBs significantly limit the primase and polymerase activities of PrimPol (negative regulatory role). PrimPol is a highly mutagenic polymerase with error specificity biased towards insertion-deletion errors. |
Co-IP (RPA, mtSSB interactions in vivo), NMR spectroscopy (RPA70 binding domain mapping), forward mutation assays, PCNA interaction tests |
Nucleic acids research |
High |
25550423
|
| 2014 |
The high myopia-associated PrimPol mutation Y89D causes a striking decrease in primase and polymerase activities, reduced affinities for DNA and nucleotides, diminished catalytic efficiency, altered structure/stability, reduced cell viability after DNA damage, and significantly slower replication fork rates in vivo. |
In vitro primase/polymerase assays, DNA/nucleotide binding assays (Kd measurements), structural analysis, cell viability assays, DNA fiber analysis |
Nucleic acids research |
High |
25262353
|
| 2015 |
PrimPol plays a crucial role in bypassing leading-strand G-quadruplex (G4) structures during DNA replication. PrimPol is unable to directly replicate G4s but can bind and reprime downstream of these structures. Disruption of either catalytic activity or the zinc finger of PrimPol results in extreme G4-dependent epigenetic instability in avian DT40 cells, indicating extensive helicase-polymerase uncoupling. |
PrimPol−/− DT40 cell knockout, epigenetic stability assays (BU-1 locus), complementation with catalytic and zinc-finger mutants, DNA replication assays |
Molecular cell |
High |
26626482
|
| 2015 |
Rad51 controls the elongation of UV-damaged DNA in a manner distinct from TLS polymerase Polη. In Rad51-depleted cells, excessive elongation of nascent DNA after UV irradiation requires PrimPol, a DNA polymerase with primase activity, indicating that Rad51 suppresses excessive PrimPol-mediated nascent DNA elongation after UV damage. |
siRNA knockdown of Rad51, DNA fiber analysis, epistasis with PrimPol knockdown |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
26627254
|
| 2016 |
Crystal structure of human PrimPol in ternary complex with DNA template-primer and incoming dNTP reveals that PrimPol's primase activity stems from near-complete lack of contacts to the DNA primer strand, allowing two dNTPs to bind initiation and elongation sites for first dinucleotide formation. The active-site cleft is constrained, precluding conventional translesion synthesis bypass of UV-induced lesions. |
X-ray crystallography (ternary complex structure), structural analysis |
Science advances |
High |
27819052
|
| 2016 |
PolDIP2 is a novel PrimPol binding partner that stimulates PrimPol's polymerase activity by enhancing DNA binding capacity and processivity. PolDIP2 also stimulates efficiency and error-free bypass of 8-oxoG lesions by PrimPol. PolDIP2 binds to PrimPol's catalytic domain. Depletion of PolDIP2 in human cells causes decreased replication fork rates similar to PrimPol−/− cells, and depletion in PrimPol−/− cells produces no further decrease. |
Co-IP (PrimPol–PolDIP2 interaction), in vitro polymerase/lesion bypass assays, domain binding mapping, DNA fiber analysis, siRNA knockdown, epistasis |
Nucleic acids research |
High |
26984527
|
| 2016 |
PrimPol's primase (repriming) activity, rather than its TLS polymerase activity, is pivotal for DNA damage tolerance. Polymerase-defective but not primase-deficient PrimPol suppresses hypersensitivity of PrimPol−/− cells to various DNA damaging agents. PrimPol reprimes closely coupled downstream of chain-terminating nucleoside analogs (CTNAs) and oxidative damage in vitro. |
Separation-of-function mutant complementation in PrimPol−/− avian cells, in vitro repriming assays, sensitivity assays to multiple genotoxins and CTNAs |
Cell cycle (Georgetown, Tex.) |
High |
27230014
|
| 2017 |
PrimPol possesses two RPA-binding motifs (identified by biophysical and crystallographic approaches). One of these motifs is critical for recruitment of PrimPol to stalled replication forks in vivo. RPA stimulates the primase activity of PrimPol. |
Crystallography (RPA-binding motif structure), biophysical binding assays, site-directed mutagenesis of RPA-binding motifs, in vivo recruitment assays at stalled forks, in vitro primase stimulation assays |
Nature communications |
High |
28534480
|
| 2017 |
PrimPol is required for replication reinitiation after mtDNA damage in vivo and in vitro. PrimPol can reinitiate stalled mtDNA replication and prime mtDNA replication from non-conventional origins. |
In vivo mtDNA replication assays, in vitro mitochondrial replication reconstitution, PrimPol-deficient cells |
Proceedings of the National Academy of Sciences of the United States of America |
High |
29073063
|
| 2018 |
The Zn-finger domain (ZnFD) of PrimPol is required for stabilizing the initiating 5'-nucleotide during primer synthesis: ZnFD is dispensable for binary complex (ssDNA binding) and pre-ternary complex (3'-nucleotide binding) formation but essential for binding/selecting the 5'-initiating nucleotide, likely interacting with the γ-phosphate moiety. ZnFD also contributes to recognizing the preferred priming sequence 3'GTC5' and to translocation/elongation during primer synthesis. |
Biochemical substrate-binding assays (EMSA), in vitro primase assays with ZnFD deletion mutants, nucleotide selection experiments |
Nucleic acids research |
High |
29608762
|
| 2018 |
PrimPol-dependent repriming limits R-loop formation during S phase. Absence of PrimPol leads to significantly increased R-loop formation around replication-blocking repeat sequences (including G-quadruplex and H-DNA motifs) across the genome during S phase in both avian and human cells. |
R-loop detection (S9.6 immunofluorescence, DRIP), PrimPol−/− cell lines, genome-wide analysis, in vivo replication assays |
The EMBO journal |
High |
30478192
|
| 2019 |
Increased PRIMPOL expression and chromatin loading, regulated by ATR activity, mediates an adaptive response in BRCA1-deficient cancer cells exposed to repeated cisplatin doses. PRIMPOL rescues fork degradation by reinitiating DNA synthesis past lesions, leading to ssDNA gaps while suppressing fork reversal. |
Electron microscopy of replication intermediates, DNA fiber analysis, PRIMPOL overexpression/knockdown, ATR inhibition, SMARCAL1 KO epistasis |
Molecular cell |
High |
31676232
|
| 2019 |
The deubiquitinase USP36 interacts with PrimPol and deubiquitinates K29-linked polyubiquitination of PrimPol, increasing its protein stability. Depletion of USP36 results in replication stress-related defects and elevated chemosensitivity. |
Co-IP (USP36–PrimPol interaction), ubiquitination assays, proteasome inhibitor experiments, siRNA knockdown |
Nucleic acids research |
Medium |
33237263
|
| 2019 |
The invariant glutamate (Glu116) in PrimPol's DxE motif (Motif A) is a critical metal ligand enhancing distinctive Mn2+-dependent reactions including error-prone TLS at 8-oxodG, TLS via primer/template realignments, and primase activity. Glu116 contributes to optimal incoming nucleotide stabilization, especially required during primer synthesis. |
Site-directed mutagenesis (D114A, E116A/D, D280A), in vitro primase and polymerase assays with Mg2+/Mn2+, EMSA (pre-ternary complex) |
DNA repair |
High |
30889508
|
| 2019 |
The cancer-associated mutation Y100H in PrimPol disables the steric gate (Tyr100) for sugar discrimination. The Y100H mutation profoundly stimulates NTP (ribonucleotide) incorporation by PrimPol, with efficiency similar to dNTP incorporation during primase and polymerase reactions in vitro. Expression of Y100H in cells causes enhanced resistance to hydroxyurea (which depletes dNTP pools). |
In silico structural modeling, site-directed mutagenesis (Y100H), in vitro NTP/dNTP incorporation assays, cellular HU resistance assays |
Scientific reports |
High |
30718533
|
| 2020 |
HR induced by bulky DNA adducts (BPDE) in mammalian cells predominantly occurs at post-replicative gaps formed by PrimPol re-priming, not at stalled forks. RAD51 recruitment under these conditions requires PrimPol-mediated gaps and resection by MRE11 and EXO1. PrimPol promotes sister chromatid exchange and genetic recombination at bulky adducts. |
PrimPol KO/knockdown, RAD51 focus formation assays, sister chromatid exchange assays, MRE11/EXO1 epistasis, DNA fiber analysis |
Nature communications |
High |
33203852
|
| 2021 |
BRCA2 associates with the essential DNA replication factor MCM10, and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation after DNA damage, while having no impact on stability of stalled replication forks. |
Co-IP (BRCA2–MCM10 interaction), DNA fiber analysis, PRIMPOL knockdown epistasis, ssDNA gap detection |
Nature communications |
High |
34645815
|
| 2021 |
PRIMPOL repriming generates ssDNA gaps that are filled by temporally distinct post-replicative mechanisms: in G2, gap filling depends on RAD18 E3 ubiquitin ligase, PCNA monoubiquitination, and REV1–POLζ TLS polymerases; in S phase, UBC13, RAD51 recombinase, and REV1–POLζ are responsible. BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity. |
DNA fiber gap-filling assay, siRNA knockdown of pathway components, cell-cycle synchronization, epistasis analysis |
Molecular cell |
High |
34624216
|
| 2021 |
BRCA1/2-deficient cells accumulate ssDNA gaps and spontaneous mutations during unperturbed DNA replication due to PRIMPOL repriming. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of RAD18 or inhibition of REV1–Polζ. REV1–Polζ protects viability of BRCA1/2-deficient cells by mutagenic repair of PRIMPOL-generated gaps. |
PRIMPOL KO/knockdown, ssDNA gap detection (S1 nuclease assay), SMUG1 epistasis, REV1-Polζ inhibitor (JH-RE-06), mutation rate analysis, xenograft models |
Molecular cell |
High |
34508659
|
| 2021 |
PrimPol-mediated repriming strictly requires repriming events downstream of ICLs for ICL traverse by a single replication fork. Recruitment of PrimPol to ICL vicinities depends on its interaction with RPA but not on FANCM translocase or the BTR complex. PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. |
Electron microscopy of replication intermediates, PRIMPOL KO cells and mice, drug sensitivity assays, RPA interaction mutants, epistasis with FANCM/BTR |
The EMBO journal |
High |
34128550
|
| 2021 |
Crystal structures of PrimPol insertion complexes with DNA template-primer and correct dCTP or erroneous dATP opposite 8-oxoG, plus extension complexes, reveal that PrimPol accommodates 8-oxoG(anti) in the active site without perturbation during correct dCMP insertion and extension, explaining predominantly error-free bypass of 8-oxoG. |
X-ray crystallography (multiple ternary/extension complexes with 8-oxoG lesion) |
Nature communications |
High |
34188055
|
| 2021 |
PLK1 phosphorylates PrimPol at a conserved residue between its two RPA-binding motifs. This phosphorylation is differentially modified throughout the cell cycle and prevents aberrant chromatin recruitment of PrimPol. Phosphorylation can be delayed and reversed in response to replication stress. Absence of PLK1-dependent PrimPol regulation induces chromosome breaks, micronuclei, and decreased survival after camptothecin, olaparib, and UV-C treatment. |
In vitro kinase assays (PLK1 phosphorylation of PrimPol), phospho-specific mutants, chromatin fractionation, cell cycle analysis, cellular survival assays |
Science advances |
High |
34860556
|
| 2021 |
PolDIP2 uses a unique arginine cluster in its C-terminal ApaG-like domain to interact with a flexible loop of PrimPol, enhancing processivity by increasing both primer-template and dNTP binding affinities of PrimPol, thereby enhancing nucleotide incorporation efficiency. |
Binding affinity measurements, in vitro polymerase processivity assays, mutagenesis of PolDIP2 arginine cluster, domain mapping |
Nucleic acids research |
High |
33533925
|
| 2022 |
CHK1 phosphorylates PRIMPOL to promote repriming irrespective of the type of replication stress, and this phosphorylation is important for cellular resistance to DNA damage. PRIMPOL-dependent repriming comes at the expense of single-strand gap formation, and constitutive PRIMPOL activity results in reduced cell fitness. |
In vitro kinase assays (CHK1 phosphorylation of PRIMPOL at Ser255), phospho-mutant complementation, DNA fiber analysis, CLASPIN overexpression to increase CHK1 signaling |
Science advances |
High |
35353580
|
| 2022 |
Pol ι deficiency unleashes PrimPol-dependent repriming, accelerating DNA replication in a pathway epistatic with ZRANB3 knockdown. This TLS-independent function of Pol ι requires its PCNA-interacting domain but not its polymerase domain, indicating Pol ι restrains PrimPol activity to prevent chromosome instability. |
Pol ι knockdown/knockout, DNA fiber analysis, ZRANB3 epistasis, PCNA-binding domain mutant of Pol ι, chromosome instability assays |
Science advances |
High |
37058556
|
| 2023 |
Nuclear actin polymerization limits PrimPol chromatin loading; chemically or genetically impairing nuclear actin polymerization leads to deregulated PrimPol chromatin loading, promoting unrestrained and discontinuous DNA synthesis and limiting RAD51 and SMARCAL1 recruitment to nascent DNA. Chromosomal instability induced by defective nuclear actin polymerization upon mild replication stress is PRIMPOL-dependent. |
Nuclear actin live imaging, chemical/genetic actin polymerization inhibition, PrimPol chromatin fractionation, DNA fiber analysis, RAD51/SMARCAL1 iPOND, PRIMPOL KO epistasis |
Nature communications |
High |
38016948
|
| 2023 |
ATR/CHK1 pathway is required for PRIMPOL-dependent repriming under KRASG12V-induced replication stress. PrimPol is phosphorylated at Ser255 (a potential CHK1 substrate site) under KRASG12V-induced stress and promotes repriming to maintain fork progression and cell survival. PrimPol-dependent repriming generates ssDNA gaps at heterochromatin, leading to genomic instability. |
KRASG12V induction, DNA fiber analysis, PrimPol phosphorylation detection, CHK1 inhibition, ATR overexpression, ssDNA gap detection |
Nature communications |
Medium |
37591859
|
| 2023 |
PRIMPOL generates ssDNA gaps in response to APOBEC3A-induced replication stress. A3A-induced ssDNA gaps are repaired by pathways involving ATR, RAD51, and translesion synthesis. PARP inhibitor and ATR inhibitor combination preferentially kills A3A-expressing cells in a PrimPol-dependent manner. |
PRIMPOL KO epistasis, ssDNA gap detection, A3A overexpression, ATR/PARP inhibitor treatment, DNA fiber analysis |
Science advances |
High |
38241374
|
| 2024 |
PRIMPOL-generated ssDNA gaps are expanded bidirectionally by MRE11 exonuclease (3'–5') and EXO1 exonuclease (5'–3'), ultimately converting gaps into DSBs. USP1 deubiquitinase promotes gap accumulation during S phase and their expansion by MRE11 and EXO1 through PCNA deubiquitination; PCNA ubiquitination prevents gap accumulation during replication. |
PRIMPOL overexpression, S1 nuclease gap assay, MRE11/EXO1 knockdown, USP1 knockdown, PCNA ubiquitination mutants, DSB detection (γ-H2AX) |
Nucleic acids research |
High |
38180818
|
| 2024 |
The CST complex (CTC1/STN1/TEN1) restricts excessive PrimPol repriming upon UV-induced replication stress. STN1 depletion stimulates p21-PrimPol interaction and facilitates PrimPol recruitment to stalled forks. p21 interacts with PrimPol and is required for enhanced PrimPol recruitment when CST is depleted. |
STN1/CTC1 knockdown, DNA fiber analysis, PrimPol recruitment to stalled forks, Co-IP (p21–PrimPol interaction), p21 knockdown epistasis |
Nucleic acids research |
Medium |
38348929
|
| 2024 |
CAF-1 promotes efficient PrimPol localization to nascent DNA; loss of CAF-1 reduces PrimPol recruitment to replication forks and suppresses ssDNA gap formation. This role is independent of CAF-1's nucleosome deposition function but relies on its localization to replication forks. |
CAF-1 knockdown, iPOND (PrimPol nascent DNA association), ssDNA gap assays, CAF-1 nucleosome deposition mutants |
Nucleic acids research |
Medium |
39558157
|
| 2024 |
RFWD3 and PRIMPOL cooperate in a fork restart pathway: in cells lacking SLFN11, fork restart proceeds through RFWD3 and PRIMPOL to facilitate gapped DNA synthesis. SLFN11 antagonizes this pathway by disrupting recruitment of RFWD3 and PRIMPOL to stalled forks in a manner dependent on its ATPase domain. |
DNA fiber analysis, SLFN11 KO/expression, RFWD3 and PRIMPOL epistasis, super-resolution microscopy, ATPase-dead SLFN11 mutant |
Nature communications |
Medium |
41372167
|
| 2019 |
WRNIP1 and PrimPol form a complex in cells. PrimPol protein expression is reduced by WRNIP1 overexpression and increased in WRNIP1-depleted cells; this reduction is suppressed by proteasome inhibitors, indicating WRNIP1 promotes proteasomal degradation of PrimPol. |
Co-IP (WRNIP1–PrimPol complex), WRNIP1 overexpression and siRNA knockdown, proteasome inhibitor treatment |
Biological & pharmaceutical bulletin |
Medium |
31061318
|
| 2021 |
TERRA R-loops interfere with semiconservative DNA replication and induce PRIMPOL-dependent repair, which initiates DNA synthesis de novo downstream of replication obstacles at telomeres. PRIMPOL acts in parallel to break-induced replication (BIR) for telomere maintenance, and PRIMPOL depletion is synthetic lethal with BIR deficiency in ALT cancer cells. |
TERRA overexpression, PRIMPOL depletion, BIR reporter assay, synthetic lethality screen, telomere FISH |
The EMBO journal |
Medium |
40624280
|
| 2022 |
PrimPol stress-triggered repriming is required for efficient hematopoietic stem cell (HSC) amplification and bone marrow reconstitution. Stimulated HSPCs show accelerated fork progression reflecting engagement of PrimPol-dependent repriming at the expense of replication fork reversal. |
Transcriptomics, single-cell and single-molecule (DNA fiber) assays on murine bone marrow cells, competitive bone marrow transplantation with PrimPol KO mice |
Molecular cell |
Medium |
36152632
|
| 2021 |
The Arg47 and Arg76 residues in the PrimPol active site contact the DNA template and are crucial for both DNA polymerase and primase activities; R76A causes near-complete loss of catalytic activity. These residues affect the dNMP incorporation spectrum on undamaged and 8-oxoG-containing templates and are required for stable PrimPol:DNA complex formation in the presence of ATP/dNTPs. |
Site-directed mutagenesis (R47A, R76A), in vitro primase/polymerase assays, EMSA |
DNA repair |
High |
33571927
|
| 2023 |
PrimPol initiates de novo DNA synthesis in cis-orientation, with the N-terminal catalytic domain (NTD) and C-terminal domain (CTD) of the same molecule cooperating for substrate binding and catalysis. The ZnFn motif residue Arg417 is required for binding the 5'-triphosphate group stabilizing the PrimPol complex with template-primer. The NTD alone can initiate DNA synthesis, while the CTD stimulates primase activity. The RPA-binding motif in the CTD modulates PrimPol binding to DNA. |
Domain deletion mutants, biochemical primase/polymerase assays, EMSA, structural modeling |
Nucleic acids research |
High |
37326028
|
| 2023 |
PrimPol is required for cell survival following loss of Y-family polymerases REV1 and POLη in a lesion-dependent manner, and plays a broader role promoting survival of cells lacking PCNA K164-dependent post-replicative gap filling. PrimPol restricts post-replicative gap length to maximize the effectiveness of interactions between REV1-bypass and PCNA K164R-bypass damage tolerance pathways. |
Genome-wide CRISPR/Cas9 screens, genetic epistasis in non-transformed p53-proficient human cells, PRIMPOL KO |
Nucleic acids research |
Medium |
37971291
|
| 2024 |
Translesion synthesis (TLS) by Polκ and Polη occurs mainly behind restarted replication forks, dependent on PrimPol repriming: TLS polymerase recruitment to DNA adducts is adduct-specific (Polκ for BPDE, Polη for cisplatin) and depends on PrimPol. TLS deficiency results in ssDNA gap accumulation in an adduct-specific manner, and gaps are processed into DSBs. |
Proximity ligation imaging at DNA adducts, PRIMPOL KO epistasis, ssDNA gap detection (S1 nuclease), DSB detection |
Cell reports |
Medium |
40014449
|