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1.
Nucleic Acids Res ; 49(9): 4831-4847, 2021 05 21.
Article in English | MEDLINE | ID: mdl-33744934

ABSTRACT

To bypass a diverse range of fork stalling impediments encountered during genome replication, cells possess a variety of DNA damage tolerance (DDT) mechanisms including translesion synthesis, template switching, and fork reversal. These pathways function to bypass obstacles and allow efficient DNA synthesis to be maintained. In addition, lagging strand obstacles can also be circumvented by downstream priming during Okazaki fragment generation, leaving gaps to be filled post-replication. Whether repriming occurs on the leading strand has been intensely debated over the past half-century. Early studies indicated that both DNA strands were synthesised discontinuously. Although later studies suggested that leading strand synthesis was continuous, leading to the preferred semi-discontinuous replication model. However, more recently it has been established that replicative primases can perform leading strand repriming in prokaryotes. An analogous fork restart mechanism has also been identified in most eukaryotes, which possess a specialist primase called PrimPol that conducts repriming downstream of stalling lesions and structures. PrimPol also plays a more general role in maintaining efficient fork progression. Here, we review and discuss the historical evidence and recent discoveries that substantiate repriming as an intrinsic replication restart pathway for maintaining efficient genome duplication across all domains of life.


Subject(s)
DNA Replication , DNA/biosynthesis , Animals , DNA/history , DNA Damage , DNA Primase/classification , DNA Primase/physiology , DNA-Directed DNA Polymerase/physiology , Genome , History, 20th Century , Models, Genetic , Stress, Physiological/genetics
2.
Mol Cell ; 79(2): 293-303.e4, 2020 07 16.
Article in English | MEDLINE | ID: mdl-32679076

ABSTRACT

Liquid-liquid phase-separated (LLPS) states are key to compartmentalizing components in the absence of membranes; however, it is unclear whether LLPS condensates are actively and specifically organized in the subcellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB), and a motor (ParA). We show that parS and ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favored by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates, and localizes non-canonical LLPS condensates in the subcellular space.


Subject(s)
Adenosine Triphosphate/physiology , Bacterial Physiological Phenomena , Escherichia coli Proteins/physiology , Phase Transition , DNA Primase/physiology , DNA, Bacterial , Microscopy/methods , Nanoparticles , Single Molecule Imaging/methods
3.
Mol Cell ; 78(6): 1237-1251.e7, 2020 06 18.
Article in English | MEDLINE | ID: mdl-32442397

ABSTRACT

DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.


Subject(s)
DNA Replication/physiology , DNA-Binding Proteins/metabolism , DNA/biosynthesis , Transcription Factors/metabolism , Cell Line, Tumor , DNA/genetics , DNA Damage/genetics , DNA Primase/metabolism , DNA Primase/physiology , DNA Repair/genetics , DNA Replication/genetics , DNA-Binding Proteins/genetics , DNA-Directed DNA Polymerase/metabolism , DNA-Directed DNA Polymerase/physiology , HEK293 Cells , Humans , K562 Cells , Multifunctional Enzymes/metabolism , Multifunctional Enzymes/physiology , Nucleotidyltransferases/metabolism , Nucleotidyltransferases/physiology , Transcription Factors/genetics
4.
Mol Cell ; 77(3): 461-474.e9, 2020 02 06.
Article in English | MEDLINE | ID: mdl-31676232

ABSTRACT

Acute treatment with replication-stalling chemotherapeutics causes reversal of replication forks. BRCA proteins protect reversed forks from nucleolytic degradation, and their loss leads to chemosensitivity. Here, we show that fork degradation is no longer detectable in BRCA1-deficient cancer cells exposed to multiple cisplatin doses, mimicking a clinical treatment regimen. This effect depends on increased expression and chromatin loading of PRIMPOL and is regulated by ATR activity. Electron microscopy and single-molecule DNA fiber analyses reveal that PRIMPOL rescues fork degradation by reinitiating DNA synthesis past DNA lesions. PRIMPOL repriming leads to accumulation of ssDNA gaps while suppressing fork reversal. We propose that cells adapt to repeated cisplatin doses by activating PRIMPOL repriming under conditions that would otherwise promote pathological reversed fork degradation. This effect is generalizable to other conditions of impaired fork reversal (e.g., SMARCAL1 loss or PARP inhibition) and suggests a new strategy to modulate cisplatin chemosensitivity by targeting the PRIMPOL pathway.


Subject(s)
DNA Primase/metabolism , DNA Replication/drug effects , DNA-Directed DNA Polymerase/metabolism , Multifunctional Enzymes/metabolism , Ubiquitin-Protein Ligases/metabolism , Cell Line, Tumor , DNA/genetics , DNA Damage/genetics , DNA Damage/physiology , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Primase/physiology , DNA Replication/genetics , DNA Replication/physiology , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/physiology , HEK293 Cells , Humans , Multifunctional Enzymes/physiology , Ubiquitin-Protein Ligases/genetics
5.
Cell ; 176(1-2): 154-166.e13, 2019 01 10.
Article in English | MEDLINE | ID: mdl-30595448

ABSTRACT

Primases have a fundamental role in DNA replication. They synthesize a primer that is then extended by DNA polymerases. Archaeoeukaryotic primases require for synthesis a catalytic and an accessory domain, the exact contribution of the latter being unresolved. For the pRN1 archaeal primase, this domain is a 115-amino acid helix bundle domain (HBD). Our structural investigations of this small HBD by liquid- and solid-state nuclear magnetic resonance (NMR) revealed that only the HBD binds the DNA template. DNA binding becomes sequence-specific after a major allosteric change in the HBD, triggered by the binding of two nucleotide triphosphates. The spatial proximity of the two nucleotides and the DNA template in the quaternary structure of the HBD strongly suggests that this small domain brings together the substrates to prepare the first catalytic step of primer synthesis. This efficient mechanism is likely general for all archaeoeukaryotic primases.


Subject(s)
DNA Primase/metabolism , DNA Primase/physiology , DNA Primers/chemistry , Animals , Binding Sites , DNA , DNA Primase/ultrastructure , DNA Primers/metabolism , DNA Replication/physiology , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Humans , Nucleotides , Protein Conformation , Protein Structural Elements/physiology
6.
J Virol ; 92(18)2018 09 15.
Article in English | MEDLINE | ID: mdl-29976672

ABSTRACT

Herpes simplex virus 1 (HSV-1) UL51 is a phosphoprotein that functions in the final envelopment in the cytoplasm and viral cell-cell spread, leading to efficient viral replication in cell cultures. To clarify the mechanism by which UL51 is regulated in HSV-1-infected cells, we focused on the phosphorylation of UL51. Mass spectrometry analysis of purified UL51 identified five phosphorylation sites in UL51. Alanine replacement of one of the identified phosphorylation sites in UL51, serine 184 (Ser-184), but not the other identified phosphorylation sites, significantly reduced viral replication and cell-cell spread in HaCaT cells. This mutation induced membranous invaginations adjacent to the nuclear membrane, the accumulation of primary enveloped virions in the invaginations and perinuclear space, and mislocalized UL34 and UL31 in punctate structures at the nuclear membrane; however, it had no effect on final envelopment in the cytoplasm of HaCaT cells. Of note, the alanine mutation in UL51 Ser-184 significantly reduced the mortality of mice following ocular infection. Phosphomimetic mutation in UL51 Ser-184 partly restored the wild-type phenotype in cell cultures and in mice. Based on these results, we concluded that some UL51 functions are specifically regulated by phosphorylation at Ser-184 and that this regulation is critical for HSV-1 replication in cell cultures and pathogenicity in vivoIMPORTANCE HSV-1 UL51 is conserved in all members of the Herpesviridae family. This viral protein is phosphorylated and functions in viral cell-cell spread and cytoplasmic virion maturation in HSV-1-infected cells. Although the downstream effects of HSV-1 UL51 have been clarified, there is a lack of information on how this viral protein is regulated as well as the significance of the phosphorylation of this protein in HSV-1-infected cells. In this study, we show that the phosphorylation of UL51 at Ser-184 promotes viral replication, cell-cell spread, and nuclear egress in cell cultures and viral pathogenicity in mice. This is the first report to identify the mechanism by which UL51 is regulated as well as the significance of UL51 phosphorylation in HSV-1 infection. Our study may provide insights into the regulatory mechanisms of other herpesviral UL51 homologs.


Subject(s)
DNA Helicases/chemistry , DNA Helicases/physiology , DNA Primase/chemistry , DNA Primase/physiology , Herpesvirus 1, Human/pathogenicity , Viral Proteins/chemistry , Viral Proteins/physiology , Virus Release , Virus Replication , Active Transport, Cell Nucleus , Animals , Cell Line , Chlorocebus aethiops , DNA Helicases/genetics , DNA Helicases/isolation & purification , DNA Primase/genetics , DNA Primase/isolation & purification , Eye/virology , HEK293 Cells , Herpes Simplex/virology , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/physiology , Humans , Mice , Phosphorylation , Protein Serine-Threonine Kinases , Vero Cells , Viral Proteins/genetics , Viral Proteins/isolation & purification , Virion/physiology , Virulence , Virus Assembly
7.
Proc Natl Acad Sci U S A ; 115(26): 6697-6702, 2018 06 26.
Article in English | MEDLINE | ID: mdl-29891690

ABSTRACT

The cellular replicative DNA polymerases cannot initiate DNA synthesis without a priming 3' OH. During DNA replication, this is supplied in the context of a short RNA primer molecule synthesized by DNA primase. The primase of archaea and eukaryotes, despite having varying subunit compositions, share sequence and structural homology. Intriguingly, archaeal primase has been demonstrated to possess the ability to synthesize DNA de novo, a property shared with the eukaryotic PrimPol enzymes. The dual RNA and DNA synthetic capabilities of the archaeal DNA primase have led to the proposal that there may be a sequential hand-off between these synthetic modes of primase. In the current work, we dissect the functional interplay between DNA and RNA synthetic modes of primase. In addition, we determine the key determinants that govern primer length definition by the archaeal primase. Our results indicate a primer measuring system that functions akin to a caliper.


Subject(s)
Archaeal Proteins/physiology , DNA Primase/physiology , DNA Primers/chemistry , Sulfolobus solfataricus/enzymology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Crystallography, X-Ray , DNA Primase/chemistry , Fluorescence Polarization , Models, Molecular , Molecular Weight , Protein Conformation , Protein Subunits
8.
Adv Exp Med Biol ; 1042: 117-133, 2017.
Article in English | MEDLINE | ID: mdl-29357056

ABSTRACT

This chapter focuses on the enzymes and mechanisms involved in lagging-strand DNA replication in eukaryotic cells. Recent structural and biochemical progress with DNA polymerase α-primase (Pol α) provides insights how each of the millions of Okazaki fragments in a mammalian cell is primed by the primase subunit and further extended by its polymerase subunit. Rapid kinetic studies of Okazaki fragment elongation by Pol δ illuminate events when the polymerase encounters the double-stranded RNA-DNA block of the preceding Okazaki fragment. This block acts as a progressive molecular break that provides both time and opportunity for the flap endonuclease 1 (FEN1) to access the nascent flap and cut it. The iterative action of Pol δ and FEN1 is coordinated by the replication clamp PCNA and produces a regulated degradation of the RNA primer, thereby preventing the formation of long-strand displacement flaps. Occasional long flaps are further processed by backup nucleases including Dna2.


Subject(s)
DNA Replication/physiology , DNA/genetics , DNA/metabolism , Eukaryota/genetics , Eukaryotic Cells/metabolism , Animals , DNA Polymerase I/metabolism , DNA Polymerase I/physiology , DNA Primase/metabolism , DNA Primase/physiology , DNA Primers/genetics , DNA Primers/metabolism , Humans , Kinetics , RNA/metabolism
9.
Nucleic Acids Res ; 44(10): 4734-44, 2016 06 02.
Article in English | MEDLINE | ID: mdl-26926109

ABSTRACT

PrimPol is a DNA damage tolerant polymerase displaying both translesion synthesis (TLS) and (re)-priming properties. This led us to study the consequences of a PrimPol deficiency in tolerating mutagenic lesions induced by members of the APOBEC/AID family of cytosine deaminases. Interestingly, during somatic hypermutation, PrimPol counteracts the generation of C>G transversions on the leading strand. Independently, mutation analyses in human invasive breast cancer confirmed a pro-mutagenic activity of APOBEC3B and revealed a genome-wide anti-mutagenic activity of PRIMPOL as well as most Y-family TLS polymerases. PRIMPOL especially prevents APOBEC3B targeted cytosine mutations within TpC dinucleotides. As C transversions induced by APOBEC/AID family members depend on the formation of AP-sites, we propose that PrimPol reprimes preferentially downstream of AP-sites on the leading strand, to prohibit error-prone TLS and simultaneously stimulate error-free homology directed repair. These in vivo studies are the first demonstrating a critical anti-mutagenic activity of PrimPol in genome maintenance.


Subject(s)
Cytidine Deaminase/metabolism , DNA Primase/physiology , DNA-Directed DNA Polymerase/physiology , Minor Histocompatibility Antigens/metabolism , Multifunctional Enzymes/physiology , Mutagenesis , Animals , B-Lymphocytes/enzymology , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , CRISPR-Cas Systems , Cell Line , Cell Survival/radiation effects , Cells, Cultured , Cytidine Deaminase/antagonists & inhibitors , DNA/metabolism , DNA Replication , Female , Humans , Immunoglobulin Class Switching , Mice, Inbred C57BL , Somatic Hypermutation, Immunoglobulin , T-Lymphocytes/enzymology , Ultraviolet Rays
10.
Cell Cycle ; 15(7): 908-18, 2016.
Article in English | MEDLINE | ID: mdl-26694751

ABSTRACT

PrimPol is a recently identified member of the archaeo-eukaryote primase (AEP) family of primase-polymerases. It has been shown that this mitochondrial and nuclear localized enzyme plays roles in the maintenance of both unperturbed replication fork progression and in the bypass of lesions after DNA damage. Here, we utilized an avian (DT40) knockout cell line to further study the consequences of loss of PrimPol (PrimPol(-/-)) on the downstream maintenance of cells after UV damage. We report that PrimPol(-/-) cells are more sensitive to UV-C irradiation in colony survival assays than Pol η-deficient cells. Although this increased UV sensitivity is not evident in cell viability assays, we show that this discrepancy is due to an enhanced checkpoint arrest after UV-C damage in the absence of PrimPol. PrimPol(-/-) arrested cells become stalled in G2, where they are protected from UV-induced cell death. Despite lacking an enzyme required for the bypass and maintenance of replication fork progression in the presence of UV damage, we show that PrimPol(-/-) cells actually have an advantage in the presence of a Chk1 inhibitor due to their slow progression through S-phase.


Subject(s)
DNA Damage , DNA Primase/physiology , DNA-Directed DNA Polymerase/physiology , G2 Phase Cell Cycle Checkpoints , Ultraviolet Rays , Animals , Cell Death , Cell Line , Cell Proliferation , Cell Survival/radiation effects , Checkpoint Kinase 1 , Chickens , DNA Primase/genetics , DNA-Directed DNA Polymerase/genetics , G2 Phase/radiation effects , Gene Knockout Techniques , Mitosis/radiation effects , p38 Mitogen-Activated Protein Kinases/physiology
11.
Proc Natl Acad Sci U S A ; 112(51): E7055-64, 2015 Dec 22.
Article in English | MEDLINE | ID: mdl-26647183

ABSTRACT

The segregation of DNA before cell division is essential for faithful genetic inheritance. In many bacteria, segregation of low-copy number plasmids involves an active partition system composed of a nonspecific DNA-binding ATPase, ParA, and its stimulator protein ParB. The ParA/ParB system drives directed and persistent movement of DNA cargo both in vivo and in vitro. Filament-based models akin to actin/microtubule-driven motility were proposed for plasmid segregation mediated by ParA. Recent experiments challenge this view and suggest that ParA/ParB system motility is driven by a diffusion ratchet mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. Here, we develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian ratchet, which effectively couples the ATPase-dependent cycling of ParA-nucleoid affinity to the motion of the ParB-bound cargo. Paradoxically, this resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work thus sheds light on an emergent phenomenon in which nonmotor proteins work collectively via mechanochemical coupling to propel cargos-an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria.


Subject(s)
DNA Primase/physiology , Escherichia coli Proteins/physiology , Models, Biological , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Escherichia coli/genetics , Escherichia coli/physiology , Mechanotransduction, Cellular/genetics , Mechanotransduction, Cellular/physiology , Movement/physiology , Plasmids/genetics , Plasmids/metabolism
12.
Proc Natl Acad Sci U S A ; 112(48): E6624-33, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26627254

ABSTRACT

After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.


Subject(s)
DNA Breaks, Double-Stranded , DNA Primase/physiology , DNA-Binding Proteins/physiology , DNA-Directed DNA Polymerase/physiology , DNA/radiation effects , Multifunctional Enzymes/physiology , Rad51 Recombinase/physiology , Ultraviolet Rays , Cell Cycle , Cell Death , Cell Line, Tumor , Cell Survival , DNA Repair , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Disease Progression , Dose-Response Relationship, Radiation , HeLa Cells , Humans , MRE11 Homologue Protein , RNA, Small Interfering/metabolism
13.
Nucleic Acids Res ; 42(9): 5830-45, 2014 May.
Article in English | MEDLINE | ID: mdl-24682820

ABSTRACT

PrimPol is a primase-polymerase involved in nuclear and mitochondrial DNA replication in eukaryotic cells. Although PrimPol is predicted to possess an archaeo-eukaryotic primase and a UL52-like zinc finger domain, the role of these domains has not been established. Here, we report that the proposed zinc finger domain of human PrimPol binds zinc ions and is essential for maintaining primase activity. Although apparently dispensable for its polymerase activity, the zinc finger also regulates the processivity and fidelity of PrimPol's extension activities. When the zinc finger is disrupted, PrimPol becomes more promutagenic, has an altered translesion synthesis spectrum and is capable of faithfully bypassing cyclobutane pyrimidine dimer photolesions. PrimPol's polymerase domain binds to both single- and double-stranded DNA, whilst the zinc finger domain binds only to single-stranded DNA. We additionally report that although PrimPol's primase activity is required to restore wild-type replication fork rates in irradiated PrimPol-/- cells, polymerase activity is sufficient to maintain regular replisome progression in unperturbed cells. Together, these findings provide the first analysis of the molecular architecture of PrimPol, describing the activities associated with, and interplay between, its functional domains and defining the requirement for its primase and polymerase activities during nuclear DNA replication.


Subject(s)
DNA Primase/chemistry , DNA-Directed DNA Polymerase/chemistry , Multifunctional Enzymes/chemistry , Animals , Catalytic Domain , Cell Line , DNA Primase/physiology , DNA Repair , DNA Replication , DNA-Directed DNA Polymerase/physiology , Electrophoretic Mobility Shift Assay , Humans , Manganese/chemistry , Multifunctional Enzymes/physiology , Protein Binding , Xenopus Proteins/chemistry , Zinc/chemistry
14.
Nat Struct Mol Biol ; 20(12): 1348-50, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24304914

ABSTRACT

Faithful bypass of replication forks encountering obstructive DNA lesions is essential to prevent fork collapse and cell death. PrimPol is a new human primase and translesion polymerase that is able to bypass fork-blocking UV-induced lesions and to restart replication by origin-independent repriming.


Subject(s)
DNA Primase/physiology , DNA Replication/physiology , DNA-Directed DNA Polymerase/physiology , Multifunctional Enzymes/physiology , Humans
15.
Mol Cell ; 52(4): 541-53, 2013 Nov 21.
Article in English | MEDLINE | ID: mdl-24207056

ABSTRACT

We describe a second primase in human cells, PrimPol, which has the ability to start DNA chains with deoxynucleotides unlike regular primases, which use exclusively ribonucleotides. Moreover, PrimPol is also a DNA polymerase tailored to bypass the most common oxidative lesions in DNA, such as abasic sites and 8-oxoguanine. Subcellular fractionation and immunodetection studies indicated that PrimPol is present in both nuclear and mitochondrial DNA compartments. PrimPol activity is detectable in mitochondrial lysates from human and mouse cells but is absent from mitochondria derived from PRIMPOL knockout mice. PRIMPOL gene silencing or ablation in human and mouse cells impaired mitochondrial DNA replication. On the basis of the synergy observed with replicative DNA polymerases Polγ and Polε, PrimPol is proposed to facilitate replication fork progression by acting as a translesion DNA polymerase or as a specific DNA primase reinitiating downstream of lesions that block synthesis during both mitochondrial and nuclear DNA replication.


Subject(s)
DNA Primase/physiology , DNA Replication , DNA-Directed DNA Polymerase/physiology , Multifunctional Enzymes/physiology , Amino Acid Sequence , Animals , Apurinic Acid/chemistry , Base Sequence , Catalytic Domain , Cell Nucleus/enzymology , DNA Polymerase II/chemistry , DNA Polymerase gamma , DNA Primase/chemistry , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA-Directed DNA Polymerase/chemistry , Deoxyadenosines/chemistry , Deoxyribonucleotides/chemistry , HEK293 Cells , HeLa Cells , Humans , Mice , Mice, Knockout , Mitochondria/enzymology , Molecular Sequence Data , Multifunctional Enzymes/chemistry
16.
Nat Struct Mol Biol ; 20(12): 1383-9, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24240614

ABSTRACT

DNA replication forks that collapse during the process of genomic duplication lead to double-strand breaks and constitute a threat to genomic stability. The risk of fork collapse is higher in the presence of replication inhibitors or after UV irradiation, which introduces specific modifications in the structure of DNA. In these cases, fork progression may be facilitated by error-prone translesion synthesis (TLS) DNA polymerases. Alternatively, the replisome may skip the damaged DNA, leaving an unreplicated gap to be repaired after replication. This mechanism strictly requires a priming event downstream of the lesion. Here we show that PrimPol, a new human primase and TLS polymerase, uses its primase activity to mediate uninterrupted fork progression after UV irradiation and to reinitiate DNA synthesis after dNTP depletion. As an enzyme involved in tolerance to DNA damage, PrimPol might become a target for cancer therapy.


Subject(s)
DNA Primase/physiology , DNA Replication/physiology , DNA-Directed DNA Polymerase/physiology , Multifunctional Enzymes/physiology , DNA Breaks, Double-Stranded , DNA Damage , DNA Primase/chemistry , DNA Primase/metabolism , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/metabolism , Genomic Instability , Humans , Multifunctional Enzymes/chemistry , Multifunctional Enzymes/metabolism , RNA, Messenger/metabolism , S Phase , Ultraviolet Rays
17.
Mol Cell ; 52(4): 566-73, 2013 Nov 21.
Article in English | MEDLINE | ID: mdl-24267451

ABSTRACT

DNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) light-damaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol η-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells.


Subject(s)
Chromosomes, Human/genetics , DNA Adducts/genetics , DNA Primase/physiology , DNA Replication , DNA-Directed DNA Polymerase/physiology , Multifunctional Enzymes/physiology , Amino Acid Sequence , Animals , Cell Proliferation , Cell Survival , Chickens , DNA Adducts/chemistry , DNA Adducts/metabolism , DNA Damage , DNA Primase/chemistry , DNA, Single-Stranded/chemistry , DNA-Directed DNA Polymerase/chemistry , G2 Phase Cell Cycle Checkpoints , Gene Knockdown Techniques , HEK293 Cells , Humans , Mice , Mice, Knockout , Molecular Sequence Data , Multifunctional Enzymes/chemistry , Ultraviolet Rays , Xenopus
18.
Mol Cell ; 37(1): 90-101, 2010 Jan 15.
Article in English | MEDLINE | ID: mdl-20129058

ABSTRACT

An AAA+ ATPase, DnaC, delivers DnaB helicase at the E. coli chromosomal origin by a poorly understood process. This report shows that mutant proteins bearing alanine substitutions for two conserved arginines in a motif named box VII are defective in DNA replication, but this deficiency does not arise from impaired interactions with ATP, DnaB, or single-stranded DNA. Despite their ability to deliver DnaB to the chromosomal origin to form the prepriming complex, this intermediate is inactive. Quantitative analysis of the prepriming complex suggests that the DnaB-DnaC complex contains three DnaC monomers per DnaB hexamer and that the interaction of primase with DnaB and primer formation triggers the release of DnaC, but not the mutants, from DnaB. The interaction of primase with DnaB and the release of DnaC mark discrete events in the transition from initiation to the elongation stage of DNA replication.


Subject(s)
DNA Primase/physiology , DNA Replication/physiology , DnaB Helicases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Motifs , Arginine/chemistry , Arginine/physiology , DNA, Single-Stranded/metabolism , DnaB Helicases/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Molecular Sequence Data , Protein Interaction Mapping , Replication Origin
19.
Protein Cell ; 1(2): 198-204, 2010 Feb.
Article in English | MEDLINE | ID: mdl-21203988

ABSTRACT

During severe acute respiratory syndrome coronavirus (SARS-CoV) infection, the activity of the replication/transcription complexes (RTC) quickly peaks at 6 hours post infection (h.p.i) and then diminishes significantly in the late post-infection stages. This "down-up-down" regulation of RNA synthesis distinguishes different viral stages: primary translation, genome replication, and finally viron assembly. Regarding the nsp8 as the primase in RNA synthesis, we confirmed that the proteolysis product of the primase (nsp8) contains the globular domain (nsp8C), and indentified the resectioning site that is notably conserved in all the three groups of coronavirus. We subsequently crystallized the complex of SARS-CoV nsp8C and nsp7, and the 3-D structure of this domain revealed its capability to interfuse into the hexadecamer super-complex. This specific proteolysis may indicate one possible mechanism by which coronaviruses to switch from viral infection to genome replication and viral assembly stages.


Subject(s)
DNA Primase/physiology , RNA, Viral/biosynthesis , Severe Acute Respiratory Syndrome/virology , Severe acute respiratory syndrome-related coronavirus/physiology , Virus Replication , Amino Acid Sequence , Crystallography, X-Ray , DNA Primase/chemistry , DNA Primase/genetics , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/physiology , Molecular Sequence Data , Protein Structure, Secondary , Severe acute respiratory syndrome-related coronavirus/chemistry , Severe acute respiratory syndrome-related coronavirus/genetics , Sequence Alignment
20.
J Gen Virol ; 90(Pt 8): 1937-1942, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19403757

ABSTRACT

A variant was selected from a clinical isolate of herpes simplex virus type 1 (HSV-1) during a single passage in the presence of a helicase-primase inhibitor (HPI) at eight times the IC(50). The variant was approximately 40-fold resistant to the HPI BAY 57-1293 and it showed significantly reduced growth in tissue culture with a concomitant reduction in virulence in a murine infection model. The variant contained a single mutation (Asn342Lys) in the UL5 predicted functional helicase motif IV. The Asn342Lys mutation was transferred to a laboratory strain, PDK cl-1, and the recombinant acquired the expected resistance and reduced growth characteristics. Comparative modelling and docking studies predicted the Asn342 position to be physically distant from the HPI interaction pocket formed by UL5 and UL52 (primase). We suggest that this mutation results in steric/allosteric modification of the HPI-binding pocket, conferring an indirect resistance to the HPI. Slower growth and moderately reduced virulence suggest that this mutation might also interfere with the helicase-primase activity.


Subject(s)
Antiviral Agents/pharmacology , DNA Helicases/physiology , DNA Primase/physiology , Drug Resistance, Viral , Herpesvirus 1, Human/pathogenicity , Mutation, Missense , Pyridines/pharmacology , Thiazoles/pharmacology , Viral Proteins/physiology , Virulence Factors/physiology , Amino Acid Substitution/genetics , Animals , DNA Helicases/chemistry , DNA Helicases/genetics , DNA Primase/chemistry , DNA Primase/genetics , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/growth & development , Mice , Models, Molecular , Sulfonamides , Viral Proteins/chemistry , Viral Proteins/genetics , Virulence , Virulence Factors/chemistry , Virulence Factors/genetics
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