Unprocessed genomic uracil as a source of DNA replication stress in cancer cells.

Alterations of bases in DNA constitute a major source of genomic instability. It is believed that base alterations trigger base excision repair (BER), generating DNA repair intermediates interfering with DNA replication. Here, we show that genomic uracil, a common type of base alteration, induces DNA replication stress (RS) without being processed by BER. In the absence of uracil DNA glycosylase (UNG), genomic uracil accumulates to high levels, DNA replication forks slow down, and PrimPol-mediated repriming is enhanced, generating single-stranded gaps in nascent DNA. ATR inhibition in UNG-deficient cells blocks the repair of uracil-induced gaps, increasing replication fork collapse and cell death. Notably, a subset of cancer cells upregulates UNG2 to suppress genomic uracil and limit RS, and these cancer cells are hypersensitive to co-treatment with ATR inhibitors and drugs increasing genomic uracil. These results reveal unprocessed genomic uracil as an unexpected source of RS and a targetable vulnerability of cancer cells.

PrimPol Variant V102A with Altered Primase and Polymerase Activities.

PrimPol is a human DNA primase-polymerase which restarts DNA synthesis beyond DNA lesions and non-B DNA structures blocking replication. Disfunction of PrimPol in cells leads to slowing of DNA replication rates in mitochondria and nucleus, accumulation of chromosome aberrations, cell cycle delay, and elevated sensitivity to DNA-damaging agents. A defective PrimPol has been suggested to be associated with the development of ophthalmic diseases, elevated mitochondrial toxicity of antiviral drugs and increased cell resistance to chemotherapy. Here, we describe a rare missense PrimPol variant V102A with altered biochemical properties identified in patients suffering from ovarian and cervical cancer. The Val102 to Ala substitution dramatically reduced both the primase and DNA polymerase activities of PrimPol as well as specifically decreased its ability to incorporate ribonucleotides. Structural analysis indicates that the V102A substitution can destabilize the hydrophobic pocket adjacent to the active site, affecting dNTP binding and catalysis.

BRCA2 promotes genomic integrity and therapy resistance primarily through its role in homology-directed repair.

Tumor suppressor BRCA2 functions in homology-directed repair (HDR), the protection of stalled replication forks, and the suppression of replicative gaps, but their relative contributions to genome integrity and chemotherapy response are under scrutiny. Here, we report that mouse and human cells require a RAD51 filament stabilization motif in BRCA2 for fork protection and gap suppression but not HDR. In mice, the loss of fork protection/gap suppression does not compromise genome stability or shorten tumor latency. By contrast, HDR deficiency increases spontaneous and replication stress-induced chromosome aberrations and tumor predisposition. Unlike with HDR, fork protection/gap suppression defects are also observed in Brca2 heterozygous cells, likely due to reduced RAD51 stabilization at stalled forks/gaps. Gaps arise from PRIMPOL activity, which is associated with 5-hydroxymethyl-2'-deoxyuridine sensitivity due to the formation of SMUG1-generated abasic sites and is exacerbated by poly(ADP-ribose) polymerase (PARP) inhibition. However, HDR proficiency has the major role in mitigating sensitivity to chemotherapeutics, including PARP inhibitors.

Response of PRIMPOL-Knockout Human Lung Adenocarcinoma A549 Cells to Genotoxic Stress.

Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage tolerance. The role of PrimPol in response to the different types of DNA damage is poorly understood. We knocked out the PRIMPOL gene in the lung carcinoma A549 cell line and characterized the response of the obtained cells to the DNA damage caused by hydrogen peroxide, methyl methanesulfonate (MMS), cisplatin, bleomycin, and ionizing radiation. The PRIMPOL knockout reduced the number of proliferating cells and cells in the G2 phase after treatment with MMS and caused a more pronounced delay of the S phase in the cisplatin-treated cells. Ionizing radiation at a dose of 10 Gy significantly increased the content of apoptotic cells among the PRIMPOL-deficient cells, while the proportion of cells undergoing necroptosis increased in both parental and knockout cells at any radiation dose. The viability of PRIMPOL-deficient cells upon the hydrogen peroxide-induced oxidative stress increased compared to the control cells, as determined by the methyl tetrazolium (MTT) assay. The obtained data indicate the involvement of PRIMPOL in the modulation of adaptive cell response to various types of genotoxic stress.

Coordination of Primer Initiation Within the Catalytic Domain of Human PrimPol.

To facilitate the eukaryotic repriming pathway of DNA damage tolerance, PrimPol synthesises de novo oligonucleotide primers downstream of polymerase-stalling obstacles. These primers enable replicative polymerases to resume synthesis and ensure the timely completion of DNA replication. Initiating synthesis de novo requires the coordination of single-stranded DNA, initiating nucleotides, and metal ions within PrimPol's active site to catalyze the formation of the first phosphodiester bond. Here we examine the interactions between human PrimPol's catalytic domain, nucleotides, and DNA template during each of the various catalytic steps to determine the 'choreography' of primer synthesis, where substrates bind in an ordered manner. Our findings show that the ability of PrimPol to conduct de novo primer synthesis is underpinned by a network of stabilising interactions between the enzyme, template, and nucleotides, as we previously observed for related primase CRISPR-Associated Prim-Pol (CAPP). Together, these findings establish a detailed model for the initiation of DNA synthesis by human PrimPol, which appears highly conserved.

Regulation of Human DNA Primase-Polymerase PrimPol.

Transmission of genetic information depends on successful completion of DNA replication. Genomic DNA is subjected to damage on a daily basis. DNA lesions create obstacles for DNA polymerases and can lead to the replication blockage, formation of DNA breaks, cell cycle arrest, and apoptosis. Cells have evolutionary adapted to DNA damage by developing mechanisms allowing elimination of lesions prior to DNA replication (DNA repair) and helping to bypass lesions during DNA synthesis (DNA damage tolerance). The second group of mechanisms includes the restart of DNA synthesis at the sites of DNA damage by DNA primase-polymerase PrimPol. Human PrimPol was described in 2013. The properties and functions of this enzyme have been extensively studied in recent years, but very little is known about the regulation of PrimPol and association between the enzyme dysfunction and diseases. In this review, we described the mechanisms of human PrimPol regulation in the context of DNA replication, discussed in detail interactions of PrimPol with other proteins, and proposed possible pathways for the regulation of human PrimPol activity. The article also addresses the association of PrimPol dysfunction with human diseases.

The insertion sequence excision enhancer: A PrimPol-based primer invasion system for immobilizing transposon-transmitted antibiotic resistance genes.

Evolutionary studies often identify genes that have been exchanged between different organisms and the phrase Lateral or Horizontal Gene Transfer is often used in this context. However, they rarely provide any mechanistic information concerning how these gene transfers might have occurred. With the astonishing increase in the number of sequences in public databases over the past two or three decades, identical antibiotic resistance genes have been identified in many different sequence contexts. One explanation for this would be that genes are initially transmitted by transposons which have subsequently decayed and can no longer be detected. Here, we provide an overview of a protein, IEE (Insertion Sequence Excision Enhancer) observed to facilitate high-frequency excision of IS629 from clinically important Escherichia coli O157:H7 and subsequently shown to affect a large class of bacterial insertion sequences which all transpose using the copy-out-paste-in transposition mechanism. Excision depends on both IEE and transposase indicating association with the transposition process itself. We review genetic and biochemical data and propose that IEE immobilizes genes carried by compound transposons by removing the flanking insertion sequence (IS) copies. The biochemical activities of IEE as a primase with the capacity to recognize DNA microhomologies and the observation that its effect appears restricted to IS families which use copy-out-paste-in transposition, suggests IS deletion occurs by abortive transposition involving strand switching (primer invasion) during the copy-out step. This reinforces the proposal made for understanding the widespread phenomenon loss of ISApl1 flanking mcr-1 in the compound transposon Tn6330 which we illustrate with a detailed model. This model also provides a convincing way to explain the high levels of IEE-induced precise IS excision.

Improvement of Gene Delivery by Minimal Bacteriophage Particles.

Direct delivery of therapeutic genes is a promising approach for treating cancers and other diseases. The current human viral vectors, however, suffer from several drawbacks, including poor cell-type specificity and difficult large-scale production. The M13 phage provides an alternative vehicle for gene therapy with engineerable specificity, but the low transduction efficiency seriously limits its translational application. In this work, we discovered important factors of cells and phages that greatly influence the phage transduction. The up-regulation of PrimPol or the down-regulation of DMBT1 in cells significantly enhanced the phage transduction efficiency. Furthermore, we found that the phage transduction efficiency was inversely correlated with the phage size. By carefully reconstructing the phage origin with the gene of interest, we designed "TransPhage" with a minimal length and maximal transduction efficiency. We showed that TransPhage successfully transduced the human cells with an excellent efficiency (up to 95%) comparable to or superior to that of the adeno-associated virus vectors. Moreover, we showed that TransPhage's tropism was specific to the cells that overexpress the target antigen, whereas adeno-associated viruses (AAVs) promiscuously infected many cell types. Using TransPhage as a gene therapy vehicle, we invented an NK-cell-mediated immunotherapy in which a membrane-bound fragment crystallizable region was introduced to cancer cells. We showed in vitro that the cancer cells expressing the membrane-bound fragment crystallizable (Fc) were effectively killed by CD16+ NK cells through an antibody-dependent cell-mediated cytotoxicity (ADCC)-like mechanism. In the xenograft mouse model, the administration of TransPhage carrying the membrane-bound Fc gene greatly suppressed tumor growth.

Spontaneous mutagenesis in human cells is controlled by REV1-Polymerase ζ and PRIMPOL.

Translesion DNA synthesis (TLS) facilitates replication over damaged or difficult-to-replicate templates by employing specialized DNA polymerases. We investigate the effect on spontaneous mutagenesis of three main TLS control mechanisms: REV1 and PCNA ubiquitylation that recruit TLS polymerases and PRIMPOL that creates post-replicative gaps. Using whole-genome sequencing of cultured human RPE-1 cell clones, we find that REV1 and Polymerase ζ are wholly responsible for one component of base substitution mutagenesis that resembles homologous recombination deficiency, whereas the remaining component that approximates oxidative mutagenesis is reduced in PRIMPOL-/- cells. Small deletions in short repeats appear in REV1-/-PCNAK164R/K164R double mutants, revealing an alternative TLS mechanism. Also, 500-5,000 bp deletions appear in REV1-/- and REV3L-/- mutants, and chromosomal instability is detectable in REV1-/-PRIMPOL-/- cells. Our results indicate that TLS protects the genome from deletions and large rearrangements at the expense of being responsible for the majority of spontaneous base substitutions.

Multi-omics analysis of DNA replication-associated primase polymerase (PRIMPOL) in pan-cancer: a potential target for prognosis and immune response.

BACKGROUND: It is critical to understand the mechanisms of human cancers in order to develop the effective anti-cancer therapeutic strategies. Recent studies indicated that primase polymerase (PRIMPOL) is strongly associated with the development of human cancers. Nevertheless, a systematic pan-cancer analysis of PRIMPOL remains to be further clarified. METHOD: Comprehensive multi-omics bioinformatics algorithms, such as TIMER2.0, GEPIA2.0 and cBioPortal, were utilized to evaluate the biological roles of PRIMPOL in pan-cancer, including the expression profiles, genomic alterations, prognostic values and immune regulation. RESULTS: PRIMPOL was upregulated in glioblastoma multiforme and kidney renal clear cell carcinoma. The brain lower grade glioma patients with enhanced PRIMPOL expression displayed poor prognostic values. We also demonstrated the PRIMPOL's immunomodulating effects on pan-cancer as well as its genomic changes and methylation levels. The aberrant expression of PRIMPOL was linked to various cancer-associated pathways, including DNA damage response, DNA repair, and angiogenesis, according to single-cell sequencing and function enrichment. CONCLUSIONS: This pan-cancer analysis offers a thorough review of the functional roles of PRIMPOL in human cancers, suggesting PRIMPOL as a potentially important biomarker for the progression and immunotherapy of various cancers.

Primase-polymerases: how to make a primer from scratch.

To pass on genetic information to the next generation, cells must faithfully replicate their genomes to provide copies for each daughter cell. To synthesise these duplicates, cells employ specialised enzymes called DNA polymerases, which rapidly and accurately replicate nucleic acid polymers. However, most polymerases lack the ability to directly initiate DNA synthesis and required specialised replicases called primases to make short polynucleotide primers, from which they then extend. Replicative primases (eukaryotes and archaea) belong to a functionally diverse enzyme superfamily known as Primase-Polymerases (Prim-Pols), with orthologues present throughout all domains of life. Characterised by a conserved catalytic Prim-Pol domain, these enzymes have evolved various roles in DNA metabolism, including DNA replication, repair, and damage tolerance. Many of these biological roles are fundamentally underpinned by the ability of Prim-Pols to generate primers de novo. This review examines our current understanding of the catalytic mechanisms utilised by Prim-Pols to initiate primer synthesis.

3'dNTP Binding Is Modulated during Primer Synthesis and Translesion by Human PrimPol.

PrimPol is a DNA primase/polymerase from the Archaeo-Eukaryotic Primase (AEP) superfamily that enables the progression of stalled replication forks by synthesizing DNA primers ahead of blocking lesions or abnormal structures in the ssDNA template. PrimPol's active site is formed by three AEP-conserved motifs: A, B and C. Motifs A and C of human PrimPol (HsPrimPol) harbor the catalytic residues (Asp114, Glu116, Asp280) acting as metal ligands, whereas motif B includes highly conserved residues (Lys165, Ser167 and His169), which are postulated to stabilize 3' incoming deoxynucleotides (dNTPs). Additionally, other putative nucleotide ligands are situated close to motif C: Lys297, almost invariant in the whole AEP superfamily, and Lys300, specifically conserved in eukaryotic PrimPols. Here, we demonstrate that His169 is absolutely essential for 3'dNTP binding and, hence, for both primase and polymerase activities of HsPrimPol, whereas Ser167 and Lys297 are crucial for the dimer synthesis initiation step during priming, but dispensable for subsequent dNTP incorporation on growing primers. Conversely, the elimination of Lys165 does not affect the overall primase function; however, it is required for damage avoidance via primer-template realignments. Finally, Lys300 is identified as an extra anchor residue to stabilize the 3' incoming dNTP. Collectively, these results demonstrate that individual ligands modulate the stabilization of 3' incoming dNTPs to optimize DNA primer synthesis efficiency during initiation and primer maturation.

Deletion of the primase-polymerases encoding gene, located in a mobile element in Thermus thermophilus HB27, leads to loss of function mutation of addAB genes.

DNA primase-polymerases (Ppol) have been shown to play active roles in DNA repair and damage tolerance, both in prokaryotes and eukaryotes. The ancestral thermophilic bacterium Thermus thermophilus strain HB27 encodes a Ppol protein among the genes present in mobile element ICETh2, absent in other T. thermophilus strains. Using different strategies we ablated the function of Ppol in HB27 cells, either by knocking out the gene through insertional mutagenesis, markerless deletion or through abolition of its catalytic activity. Whole genome sequencing of this diverse collection of Ppol mutants showed spontaneous loss of function mutation in the helicase-nuclease AddAB in every ppol mutant isolated. Given that AddAB is a major player in recombinational repair in many prokaryotes, with similar activity to the proteobacterial RecBCD complex, we have performed a detailed characterization of the ppol mutants in combination with addAB mutants. The results show that knockout addAB mutants are more sensitive to DNA damage agents than the wild type, and present a dramatic three orders of magnitude increase in natural transformation efficiencies with both plasmid and lineal DNA, whereas ppol mutants show defects in plasmid stability. Interestingly, DNA-integrity comet assays showed that the genome of all the ppol and/or addAB mutants was severely affected by widespread fragmentation, however, this did not translate in neat loss of viability of the strains. All these data support that Ppol appears to keep in balance the activity of AddAB as a part of the DNA housekeeping maintenance in T. thermophilus HB27, thus, playing a key role in its genome stability.

Stress-triggered hematopoietic stem cell proliferation relies on PrimPol-mediated repriming.

Stem cell division is linked to tumorigenesis by yet-elusive mechanisms. The hematopoietic system reacts to stress by triggering hematopoietic stem and progenitor cell (HSPC) proliferation, which can be accompanied by chromosomal breakage in activated hematopoietic stem cells (HSCs). However, whether these lesions persist in their downstream progeny and induce a canonical DNA damage response (DDR) remains unclear. Inducing HSPC proliferation by simulated viral infection, we report that the associated DNA damage is restricted to HSCs and that proliferating HSCs rewire their DDR upon endogenous and clastogen-induced damage. Combining transcriptomics, single-cell and single-molecule assays on murine bone marrow cells, we found accelerated fork progression in stimulated HSPCs, reflecting engagement of PrimPol-dependent repriming, at the expense of replication fork reversal. Ultimately, competitive bone marrow transplantation revealed the requirement of PrimPol for efficient HSC amplification and bone marrow reconstitution. Hence, fine-tuning replication fork plasticity is essential to support stem cell functionality upon proliferation stimuli.

MDM2 binds and ubiquitinates PARP1 to enhance DNA replication fork progression.

The MDM2 oncoprotein antagonizes the tumor suppressor p53 by physical interaction and ubiquitination. However, it also sustains the progression of DNA replication forks, even in the absence of functional p53. Here, we show that MDM2 binds, inhibits, ubiquitinates, and destabilizes poly(ADP-ribose) polymerase 1 (PARP1). When cellular MDM2 levels are increased, this leads to accelerated progression of DNA replication forks, much like pharmacological inhibition of PARP1. Conversely, overexpressed PARP1 restores normal fork progression despite elevated MDM2. Strikingly, MDM2 profoundly reduces the frequency of fork reversal, revealed as four-way junctions through electron microscopy. Depletion of RECQ1 or the primase/polymerase (PRIMPOL) reverses the MDM2-mediated acceleration of the nascent DNA elongation rate. MDM2 also increases the occurrence of micronuclei, and it exacerbates camptothecin-induced cell death. In conclusion, high MDM2 levels phenocopy PARP inhibition in modulation of fork restart, representing a potential vulnerability of cancer cells.

Functional Domain Mapping of Werner Interacting Protein 1 (WRNIP1).

Werner helicase-interacting protein 1 (WRNIP1) belongs to the AAA+ ATPase family and is conserved from Escherichia coli to human. In addition to an ATPase domain in the middle region of WRNIP1, WRNIP1 contains a ubiquitin-binding zinc-finger (UBZ) domain and two leucine zipper motifs in the N-terminal and C-terminal regions, respectively. Here, we report that the UBZ domain of WRNIP1 is responsible for the reduced levels of UV-induced proliferating cell nuclear antigen (PCNA) monoubiquitylation in POLH-disrupted (polymerase η (Polη)-deficient) cells, and that the ATPase domain of WRNIP1 is involved in regulating the level of the PrimPol protein. The suppression of UV sensitivity of Polη-deficient cells by deletion of WRNIP1 was abolished by expression of the mutant WRNIP1 lacking the UBZ domain or ATPase domain, but not by the mutant lacking the leucine zipper domain in WRNIP1/POLH double-disrupted cells. The leucine zipper domain of WRNIP1 was required for its interaction with RAD18, a key factor in TLS (DNA translesion synthesis), and DNA polymerase δ catalytic subunit, POLD1. On the basis of these findings, we discuss the possible role of WRNIP1 in TLS.

PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy.

DNA replication can encounter blocking obstacles, leading to replication stress and genome instability. There are several mechanisms for evading this blockade. One mechanism consists of repriming ahead of the obstacles, creating a new starting point; in humans, PrimPol is responsible for carrying out this task. PrimPol is a primase that operates in both the nucleus and mitochondria. In contrast with conventional primases, PrimPol is a DNA primase able to initiate DNA synthesis de novo using deoxynucleotides, discriminating against ribonucleotides. In vitro, PrimPol can act as a DNA primase, elongating primers that PrimPol itself sythesizes, or as translesion synthesis (TLS) DNA polymerase, elongating pre-existing primers across lesions. However, the lack of evidence for PrimPol polymerase activity in vivo suggests that PrimPol only acts as a DNA primase. Here, we provide a comprehensive review of human PrimPol covering its biochemical properties and structure, in vivo function and regulation, and the processes that take place to fill the gap-containing lesion that PrimPol leaves behind. Finally, we explore the available data on human PrimPol expression in different tissues in physiological conditions and its role in cancer.

PRIMPOL competes with RAD51 to resolve G-quadruplex-induced replication stress via its interaction with RPA.

PRIMPOL (primase-polymerase) is a recently discovered DNA primase-polymerase involved in DNA damage tolerance and replication stress response in eukaryotic cells. However, the detailed mechanism of the PRIMPOL response to replication stress remains elusive. Here, we demonstrate that replication-related factors, including replication protein A (RPA), regulate the accumulation of PRIMPOL in subnuclear foci in response to replication stress induced by replication inhibitors. Moreover, PRIMPOL works at G-quadruplexes (G4s) in human cells to resolve the replication stress induced by G4s. The formation of PRIMPOL foci persists throughout the cell cycle. We further demonstrate that PRIMPOL competes with RAD51 to resolve G4-induced replication stress. In conclusion, our results provide novel insight into the mechanism of PRIMPOL in G4s to resolve replication stress and competition between PRIMPOL (repriming)- and RAD51 (fork reversal)-mediated pathways, which indicates a new strategy to improve the tumor response to DNA-damaging chemotherapy by targeting the PRIMPOL pathway.

Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis.

PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting de novo synthesis of DNA primers, and especially when selecting the 3'nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg291 residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3'-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg291, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs versus ddNTPs at the 3'elongation site, thus avoiding synthesis of abortive primers.

Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells.

PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.