ABSTRACT
PARP inhibitors (PARPi), known for their ability to induce replication gaps and accelerate replication forks, have become potent agents in anticancer therapy. However, the molecular mechanism underlying PARPi-induced fork acceleration has remained elusive. Here, we show that the first PARPi-induced effect on DNA replication is an increased replication fork rate, followed by a secondary reduction in origin activity. Through the systematic knockdown of human DNA polymerases, we identify POLA1 as mediator of PARPi-induced fork acceleration. This acceleration depends on both DNA polymerase α and primase activities. Additionally, the depletion of POLA1 increases the accumulation of replication gaps induced by PARP inhibition, sensitizing cells to PARPi. BRCA1-depleted cells are especially susceptible to the formation of replication gaps under POLA1 inhibition. Accordingly, BRCA1 deficiency sensitizes cells to POLA1 inhibition. Thus, our findings establish the POLA complex as important player in PARPi-induced fork acceleration and provide evidence that lagging strand synthesis represents a targetable vulnerability in BRCA1-deficient cells.
Subject(s)
BRCA1 Protein , DNA Primase , DNA Replication , DNA, Single-Stranded , Poly(ADP-ribose) Polymerase Inhibitors , Humans , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , DNA Primase/metabolism , DNA Primase/genetics , BRCA1 Protein/metabolism , BRCA1 Protein/genetics , DNA Replication/drug effects , DNA, Single-Stranded/metabolism , DNA, Single-Stranded/genetics , DNA-Directed DNA Polymerase/metabolism , DNA-Directed DNA Polymerase/genetics , Cell Line, Tumor , DNA Polymerase IABSTRACT
G-quadruplexes (G4s) form throughout the genome and influence important cellular processes. Their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected role for the double-stranded DNA (dsDNA) translocase helicase-like transcription factor (HLTF) in responding to G4s. We show that HLTF, which is enriched at G4s in the human genome, can directly unfold G4s in vitro and uses this ATP-dependent translocase function to suppress G4 accumulation throughout the cell cycle. Additionally, MSH2 (a component of MutS heterodimers that bind G4s) and HLTF act synergistically to suppress G4 accumulation, restrict alternative lengthening of telomeres, and promote resistance to G4-stabilizing drugs. In a discrete but complementary role, HLTF restrains DNA synthesis when G4s are stabilized by suppressing primase-polymerase (PrimPol)-dependent repriming. Together, the distinct roles of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability.
Subject(s)
DNA Primase , DNA Replication , DNA-Binding Proteins , G-Quadruplexes , Genomic Instability , MutS Homolog 2 Protein , Transcription Factors , Humans , Transcription Factors/metabolism , Transcription Factors/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , MutS Homolog 2 Protein/metabolism , MutS Homolog 2 Protein/genetics , DNA Primase/metabolism , DNA Primase/genetics , Telomere Homeostasis , DNA Damage , HEK293 Cells , Multifunctional Enzymes/metabolism , Multifunctional Enzymes/genetics , DNA-Directed DNA PolymeraseABSTRACT
Telomeres at the end of eukaryotic chromosomes are extended by a specialized set of enzymes and telomere-associated proteins, collectively termed here the telomere "replisome." The telomere replisome acts on a unique replicon at each chromosomal end of the telomeres, the 3' DNA overhang. This telomere replication process is distinct from the replisome mechanism deployed to duplicate the human genome. The G-rich overhang is first extended before the complementary C-strand is filled in. This overhang is extended by telomerase, a specialized ribonucleoprotein and reverse transcriptase. The overhang extension process is terminated when telomerase is displaced by CTC1-STN1-TEN1 (CST), a single-stranded DNA-binding protein complex. CST then recruits DNA polymerase α-primase to complete the telomere replication process by filling in the complementary C-strand. In this chapter, the recent structure-function insights into the human telomere C-strand fill-in machinery (DNA polymerase α-primase and CST) will be discussed.
Subject(s)
DNA Polymerase I , DNA Primase , DNA Replication , Telomere-Binding Proteins , Telomere , Humans , Telomere/metabolism , Telomere/genetics , DNA Polymerase I/metabolism , DNA Polymerase I/genetics , DNA Polymerase I/chemistry , DNA Primase/metabolism , DNA Primase/genetics , DNA Primase/chemistry , Telomere-Binding Proteins/metabolism , Telomere-Binding Proteins/genetics , Telomerase/metabolism , Telomerase/geneticsABSTRACT
Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.
Subject(s)
DNA Polymerase I , Shelterin Complex , Telomere-Binding Proteins , Telomere , Humans , Cryoelectron Microscopy , DNA Polymerase I/metabolism , DNA Primase/metabolism , DNA Primase/genetics , Models, Molecular , Phosphorylation , Shelterin Complex/metabolism , Telomerase/metabolism , Telomere/metabolism , Telomere-Binding Proteins/metabolismABSTRACT
Replication repriming by the specialized primase-polymerase PRIMPOL ensures the continuity of DNA synthesis during replication stress. PRIMPOL activity generates residual post-replicative single-stranded nascent DNA gaps, which are linked with mutagenesis and chemosensitivity in BRCA1/2-deficient models, and which are suppressed by replication fork reversal mediated by the DNA translocases SMARCAL1 and ZRANB3. Here, we report that the MRE11 regulator MRNIP limits the prevalence of PRIMPOL and MRE11-dependent ssDNA gaps in cells in which fork reversal is perturbed either by treatment with the PARP inhibitor Olaparib, or by depletion of SMARCAL1 or ZRANB3. MRNIP-deficient cells are sensitive to PARP inhibition and accumulate PRIMPOL-dependent DNA damage, supportive of a pro-survival role for MRNIP linked to the regulation of gap prevalence. In MRNIP-deficient cells, post-replicative gap filling is driven in S-phase by UBC13-mediated template switching involving REV1 and the TLS polymerase Pol-ζ. Our findings represent the first report of modulation of post-replicative ssDNA gap dynamics by a direct MRE11 regulator.
Subject(s)
DNA Helicases , DNA Primase , DNA Replication , DNA, Single-Stranded , DNA-Directed DNA Polymerase , MRE11 Homologue Protein , DNA, Single-Stranded/metabolism , DNA, Single-Stranded/genetics , MRE11 Homologue Protein/metabolism , MRE11 Homologue Protein/genetics , Humans , DNA-Directed DNA Polymerase/metabolism , DNA-Directed DNA Polymerase/genetics , DNA Primase/metabolism , DNA Primase/genetics , DNA Helicases/metabolism , DNA Helicases/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , DNA Damage , Phthalazines/pharmacology , Piperazines/pharmacology , Multifunctional Enzymes/genetics , Multifunctional Enzymes/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Carrier Proteins/metabolism , Carrier Proteins/geneticsABSTRACT
CrAss-like phages play an important role in maintaining ecological balance in the human intestinal microbiome. However, their genetic diversity and lifestyle are still insufficiently studied. In this study, a novel CrAssE-Sib phage genome belonging to the epsilon crAss-like phage genomes was found. Comparative analysis indicated that epsilon crAss-like phages are divided into two putative genera, which were proposed to be named Epsilonunovirus and Epsilonduovirus; CrAssE-Sib belongs to the former. The crAssE-Sib genome contains a diversity-generating retroelement (DGR) cassette with all essential elements, including the reverse transcriptase (RT) and receptor binding protein (RBP) genes. However, this RT contains the GxxxSP motif in its fourth domain instead of the usual GxxxSQ motif found in all known phage and bacterial DGRs. RBP encoded by CrAssE-Sib and other Epsilonunoviruses has an unusual structure, and no similar phage proteins were found. In addition, crAssE-Sib and other Epsilonunoviruses encode conserved prophage repressor and anti-repressors that could be involved in lysogenic-to-lytic cycle switches. Notably, DNA primase sequences of epsilon crAss-like phages are not included in the monophyletic group formed by the DNA primases of all other crAss-like phages. Therefore, epsilon crAss-like phage substantially differ from other crAss-like phages, indicating the need to classify these phages into a separate family.
Subject(s)
Bacteriophages , Genome, Viral , Phylogeny , Bacteriophages/genetics , Bacteriophages/classification , Viral Proteins/genetics , Viral Proteins/metabolism , Retroelements , Genetic Variation , Prophages/genetics , DNA, Viral/genetics , DNA Primase/genetics , DNA Primase/metabolism , Genomics/methods , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/metabolismABSTRACT
Several recent cryo-electron microscopy (cryo-EM) studies about the eukaryotic primosome, including the human primosome described by Yin et al. in this issue, have uncovered the structural intricacies between the RNA primase and the DNA polymerase. These studies show that these two partners tango on DNA to synthesize a hybrid primer composed of ~ 10 nucleotide (nt) RNA and ~ 10-nt DNA. They reveal key intermediate steps involved in this process; from the self-inhibited apo state to the initiation of RNA primer synthesis, RNA primer handover to the polymerase, primer elongation by polymerase, and finally, primer termination and release. Remarkably, the polymerase domain orchestrates all major steps during primer synthesis.
Subject(s)
DNA Polymerase I , DNA , RNA , Humans , Cryoelectron Microscopy , DNA/chemistry , DNA/metabolism , DNA/genetics , DNA Polymerase I/metabolism , DNA Polymerase I/chemistry , DNA Primase/metabolism , DNA Primase/chemistry , DNA Primase/genetics , DNA Primers/genetics , DNA Replication , RNA/chemistry , RNA/metabolism , RNA/geneticsABSTRACT
The ParABS system is crucial for the faithful segregation and inheritance of many bacterial chromosomes and low-copy-number plasmids. However, despite extensive research, the spatiotemporal dynamics of the ATPase ParA and its connection to the dynamics and positioning of the ParB-coated cargo have remained unclear. In this study, we utilize high-throughput imaging, quantitative data analysis, and computational modeling to explore the in vivo dynamics of ParA and its interaction with ParB-coated plasmids and the nucleoid. As previously observed, we find that F-plasmid ParA undergoes collective migrations ("flips") between cell halves multiple times per cell cycle. We reveal that a constricting nucleoid is required for these migrations and that they are triggered by a plasmid crossing into the cell half with greater ParA. Using simulations, we show that these dynamics can be explained by the combination of nucleoid constriction and cooperative ParA binding to the DNA, in line with the behavior of other ParA proteins. We further show that these ParA flips act to equally partition plasmids between the two lobes of the constricted nucleoid and are therefore important for plasmid stability, especially in fast growth conditions for which the nucleoid constricts early in the cell cycle. Overall, our work identifies a second mode of action of the ParABS system and deepens our understanding of how this important segregation system functions.
Subject(s)
Escherichia coli , Plasmids , Plasmids/metabolism , Plasmids/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/genetics , Chromosomes, Bacterial/metabolism , Chromosomes, Bacterial/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/genetics , Chromosome Segregation , DNA Primase/metabolism , DNA Primase/genetics , DNA, Bacterial/genetics , DNA, Bacterial/metabolismABSTRACT
The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of Xenopus laevis polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5' end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer-template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase.
Subject(s)
Cryoelectron Microscopy , DNA Polymerase I , DNA Primase , DNA Replication , Models, Molecular , Xenopus laevis , DNA Primase/chemistry , DNA Primase/metabolism , DNA Primase/genetics , DNA Polymerase I/metabolism , DNA Polymerase I/chemistry , Animals , Catalytic Domain , DNA/metabolism , DNA/chemistry , DNA/biosynthesis , DNA Primers/metabolism , DNA Primers/genetics , RNA/metabolism , RNA/chemistry , Protein ConformationABSTRACT
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.
Subject(s)
DNA Primase , DNA-Directed DNA Polymerase , Multifunctional Enzymes , Mutation, Missense , Ovarian Neoplasms , Uterine Cervical Neoplasms , Female , Humans , Amino Acid Substitution , Catalytic Domain , Crystallography, X-Ray , DNA Primase/metabolism , DNA Primase/chemistry , DNA Primase/genetics , DNA Replication , DNA-Directed DNA Polymerase/metabolism , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/chemistry , Models, Molecular , Multifunctional Enzymes/metabolism , Multifunctional Enzymes/genetics , Multifunctional Enzymes/chemistry , Protein Conformation , Uterine Cervical Neoplasms/genetics , Ovarian Neoplasms/geneticsABSTRACT
DNA replication stress, caused by various endogenous and exogenous agents, halt or stall DNA replication progression. Cells have developed diverse mechanisms to tolerate and overcome replication stress, enabling them to continue replication. One effective strategy to overcome stalled replication involves skipping the DNA lesion using a specialized polymerase known as PrimPol, which reinitiates DNA synthesis downstream of the damage. However, the mechanism regulating PrimPol repriming is largely unclear. In this study, we observe that knockdown of STN1 or CTC1, components of the CTC1/STN1/TEN1 complex, leads to enhanced replication progression following UV exposure. We find that such increased replication is dependent on PrimPol, and PrimPol recruitment to stalled forks increases upon CST depletion. Moreover, we find that p21 is upregulated in STN1-depleted cells in a p53-independent manner, and p21 depletion restores normal replication rates caused by STN1 deficiency. We identify that p21 interacts with PrimPol, and STN1 depletion stimulates p21-PrimPol interaction and facilitates PrimPol recruitment to stalled forks. Our findings reveal a previously undescribed interplay between CST, PrimPol and p21 in promoting repriming in response to stalled replication, and shed light on the regulation of PrimPol repriming at stalled forks.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21 , DNA Primase , DNA Replication , DNA-Directed DNA Polymerase , Multifunctional Enzymes , Telomere-Binding Proteins , Ultraviolet Rays , Humans , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA Primase/metabolism , DNA Primase/genetics , DNA-Directed DNA Polymerase/metabolism , DNA-Directed DNA Polymerase/genetics , Telomere-Binding Proteins/metabolism , Telomere-Binding Proteins/genetics , Multifunctional Enzymes/genetics , Multifunctional Enzymes/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , DNA DamageABSTRACT
It is well-established that, through canonical functions in transcription and DNA repair, the tumor suppressor p53 plays a central role in safeguarding cells from the consequences of DNA damage. Recent data retrieved in tumor and stem cells demonstrated that p53 also carries out non-canonical functions when interacting with the translesion synthesis (TLS) polymerase iota (POLι) at DNA replication forks. This protein complex triggers a DNA damage tolerance (DDT) mechanism controlling the DNA replication rate. Given that the levels of p53 trigger non-binary rheostat-like functions in response to stress or during differentiation, we explore the relevance of the p53 levels for its DDT functions at the fork. We show that subtle changes in p53 levels modulate the contribution of some DDT factors including POLι, POLη, POLζ, REV1, PCNA, PRIMPOL, HLTF and ZRANB3 to the DNA replication rate. Our results suggest that the levels of p53 are central to coordinate the balance between DDT pathways including (i) fork-deceleration by the ZRANB3-mediated fork reversal factor, (ii) POLι-p53-mediated fork-slowing, (iii) POLι- and POLη-mediated TLS and (iv) PRIMPOL-mediated fork-acceleration. Collectively, our study reveals the relevance of p53 protein levels for the DDT pathway choice in replicating cells.
Subject(s)
DNA Damage , DNA Polymerase iota , DNA Replication , DNA-Directed DNA Polymerase , Tumor Suppressor Protein p53 , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , DNA-Directed DNA Polymerase/metabolism , Humans , Proliferating Cell Nuclear Antigen/metabolism , Proliferating Cell Nuclear Antigen/genetics , DNA Repair , Nucleotidyltransferases/metabolism , Nucleotidyltransferases/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Multifunctional Enzymes/metabolism , Multifunctional Enzymes/genetics , DNA Primase/metabolism , DNA Primase/genetics , DNA Damage ToleranceABSTRACT
Eukaryotic DNA replication depends on the primosome - a complex of DNA polymerase alpha (Pol α) and primase - to initiate DNA synthesis by polymerisation of an RNA-DNA primer. Primer synthesis requires the tight coordination of primase and polymerase activities. Recent cryo-electron microscopy (cryoEM) analyses have elucidated the extensive conformational transitions required for RNA primer handover between primase and Pol α and primer elongation by Pol α. Because of the intrinsic flexibility of the primosome, however, structural information about the initiation of RNA primer synthesis is still lacking. Here, we capture cryoEM snapshots of the priming reaction to reveal the conformational trajectory of the human primosome that brings DNA primase subunits 1 and 2 (PRIM1 and PRIM2, respectively) together, poised for RNA synthesis. Furthermore, we provide experimental evidence for the continuous association of primase subunit PRIM2 with the RNA primer during primer synthesis, and for how both initiation and termination of RNA primer polymerisation are licenced by specific rearrangements of DNA polymerase alpha catalytic subunit (POLA1), the polymerase subunit of Pol α. Our findings fill a critical gap in our understanding of the conformational changes that underpin the synthesis of the RNA primer by the primosome. Together with existing evidence, they provide a complete description of the structural dynamics of the human primosome during DNA replication initiation.
Subject(s)
DNA Polymerase I , DNA Primase , Humans , DNA Primase/genetics , DNA Primase/metabolism , Cryoelectron Microscopy , DNA Polymerase I/genetics , RNA , DNA ReplicationABSTRACT
The Mpox pandemic, caused by the Mpox virus (or monkeypox virus, MPXV), has gained global attention. The D5 protein, a putative helicase-primase found in MPXV, plays a vital role in viral replication and genome uncoating. Here we determined multiple cryo-EM structures of full-length hexameric D5 in diverse states. These states were captured during ATP hydrolysis while moving along the single-stranded DNA (ssDNA) track. Through comprehensive structural analysis combined with the helicase activity system, we revealed that when the primase domain is truncated or the interaction between the primase and helicase domains is disrupted, the double-stranded DNA (dsDNA) unwinds into ssDNA, suggesting a critical regulatory role of the primase domain. Two transition states bound with ssDNA substrate during unwinding reveals that two ATP molecules were consumed to drive DNA moving forward two nucleotides. Collectively, our findings shed light on the molecular mechanism that links ATP hydrolysis to the DNA unwinding in poxviruses.
Subject(s)
DNA Primase , Monkeypox virus , DNA Primase/chemistry , DNA Primase/genetics , DNA Primase/metabolism , Monkeypox virus/genetics , Monkeypox virus/metabolism , DNA Helicases/metabolism , DNA/chemistry , DNA, Single-Stranded , Adenosine Triphosphate/metabolismABSTRACT
Transcription-replication conflicts (TRCs), especially Head-On TRCs (HO-TRCs) can introduce R-loops and DNA damage, however, the underlying mechanisms are still largely unclear. We previously identified a chloroplast-localized RNase H1 protein AtRNH1C that can remove R-loops and relax HO-TRCs for genome integrity. Through the mutagenesis screen, we identify a mutation in chloroplast-localized primase ATH that weakens the binding affinity of DNA template and reduces the activities of RNA primer synthesis and delivery. This slows down DNA replication, and reduces competition of transcription-replication, thus rescuing the developmental defects of atrnh1c. Strand-specific DNA damage sequencing reveals that HO-TRCs cause DNA damage at the end of the transcription unit in the lagging strand and overexpression of ATH can boost HO-TRCs and exacerbates DNA damage. Furthermore, mutation of plastid DNA polymerase Pol1A can similarly rescue the defects in atrnh1c mutants. Taken together these results illustrate a potentially conserved mechanism among organisms, of which the primase activity can promote the occurrence of transcription-replication conflicts leading to HO-TRCs and genome instability.
Subject(s)
DNA Primase , DNA Replication , DNA Primase/genetics , DNA Primase/metabolism , DNA Replication/genetics , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , DNA Damage , MutationABSTRACT
All animals must maintain genome and proteome integrity, especially when experiencing endogenous or exogenous stress. To cope, organisms have evolved sophisticated and conserved response systems: unfolded protein responses (UPRs) ensure proteostasis, while DNA damage responses (DDRs) maintain genome integrity. Emerging evidence suggests that UPRs and DDRs crosstalk, but this remains poorly understood. Here, we demonstrate that depletion of the DNA primases pri-1 or pri-2, which synthesize RNA primers at replication forks and whose inactivation causes DNA damage, activates the UPR of the endoplasmic reticulum (UPR-ER) in Caenorhabditis elegans, with especially strong activation in the germline. We observed activation of both the inositol-requiring-enzyme 1 (ire-1) and the protein kinase RNA-like endoplasmic reticulum kinase (pek-1) branches of the (UPR-ER). Interestingly, activation of the (UPR-ER) output gene heat shock protein 4 (hsp-4) was partially independent of its canonical activators, ire-1 and X-box binding protein (xbp-1), and instead required the third branch of the (UPR-ER), activating transcription factor 6 (atf-6), suggesting functional redundancy. We further found that primase depletion specifically induces the (UPR-ER), but not the distinct cytosolic or mitochondrial UPRs, suggesting that primase inactivation causes compartment-specific rather than global stress. Functionally, loss of ire-1 or pek-1 sensitizes animals to replication stress caused by hydroxyurea. Finally, transcriptome analysis of pri-1 embryos revealed several deregulated processes that could cause (UPR-ER) activation, including protein glycosylation, calcium signaling, and fatty acid desaturation. Together, our data show that the (UPR-ER), but not other UPRs, responds to replication fork stress and that the (UPR-ER) is required to alleviate this stress.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , DNA Primase/genetics , DNA Primase/metabolism , Unfolded Protein Response , Cell Cycle Proteins/genetics , DNA Damage , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress/geneticsABSTRACT
The primase/polymerase PRIMPOL restarts DNA synthesis when replication is arrested by template impediments. However, we do not have a comprehensive view of how PRIMPOL-dependent repriming integrates with the main pathways of damage tolerance, REV1-dependent 'on-the-fly' lesion bypass at the fork and PCNA ubiquitination-dependent post-replicative gap filling. Guided by genome-wide CRISPR/Cas9 screens to survey the genetic interactions of PRIMPOL in a non-transformed and p53-proficient human cell line, we find that PRIMPOL is needed for cell survival following loss of the Y-family polymerases REV1 and POLη in a lesion-dependent manner, while it plays a broader role in promoting survival of cells lacking PCNA K164-dependent post-replicative gap filling. Thus, while REV1- and PCNA K164R-bypass provide two layers of protection to ensure effective damage tolerance, PRIMPOL is required to maximise the effectiveness of the interaction between them. We propose this is through the restriction of post-replicative gap length provided by PRIMPOL-dependent repriming.
Subject(s)
DNA Damage , DNA Primase , DNA-Directed DNA Polymerase , Humans , DNA Primase/genetics , DNA Primase/metabolism , DNA Replication , Multifunctional Enzymes/genetics , Multifunctional Enzymes/metabolism , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , DNA-Directed DNA Polymerase/metabolismABSTRACT
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.
Subject(s)
Adenocarcinoma of Lung , DNA-Directed DNA Polymerase , Humans , DNA-Directed DNA Polymerase/metabolism , A549 Cells , Cisplatin/pharmacology , Hydrogen Peroxide/pharmacology , DNA Replication , DNA Damage , Adenocarcinoma of Lung/genetics , DNA Primase/genetics , DNA Primase/metabolism , Multifunctional Enzymes/genetics , Multifunctional Enzymes/metabolismABSTRACT
BACKGROUND: The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD). RESULTS: Herein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase. CONCLUSIONS: We conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.
Subject(s)
Arabidopsis , DNA Primase , Animals , DNA Primase/genetics , DNA Primase/chemistry , DNA Primase/metabolism , DNA Helicases/chemistry , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Arabidopsis/metabolism , Mitochondria/metabolism , Zinc Fingers , Ribonucleotides , DNA Replication , Bacteriophage T7/geneticsABSTRACT
Bovine herpesvirus type 1 (BoHV-1) is an important agricultural pathogen that infects cattle and other ruminants worldwide. Though it was first sequenced and annotated over twenty years ago, the Cooper strain, used in this study, was sequenced as recently as 2012 and is currently said to encode 72 unique proteins. However, tandem mass spectrometry has identified several peptides produced during active infection that align with the BoHV-1 genome in unannotated regions. One of these abundant peptides, "ORF M", aligned antisense to the DNA helicase/primase protein UL5. This study characterizes the novel transcript and its protein product and provides evidence to support the existence of homolog protein-coding genes in other Herpesviruses.