RESUMO
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.
Assuntos
Replicação do DNA , Hematopoese , Camundongos , Animais , Hematopoese/genética , Células-Tronco Hematopoéticas/fisiologia , Dano ao DNA , Proliferação de CélulasRESUMO
BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and spontaneous mutations during unperturbed DNA replication due to repriming by the DNA primase-polymerase PRIMPOL. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of the translesion synthesis (TLS) factor RAD18 or inhibition of the error-prone TLS polymerase complex REV1-Polζ by the small molecule JH-RE-06. JH-RE-06 treatment of BRCA1/2-deficient cells results in reduced mutation rates and PRIMPOL- and SMUG1-dependent loss of viability. Through cellular and animal studies, we demonstrate that JH-RE-06 is preferentially toxic toward HR-deficient cancer cells. Furthermore, JH-RE-06 remains effective toward PARP inhibitor (PARPi)-resistant BRCA1 mutant cells and displays additive toxicity with crosslinking agents or PARPi. Collectively, these studies identify a protective and mutagenic role for REV1-Polζ in BRCA1/2 mutant cells and provide the rationale for using REV1-Polζ inhibitors to treat BRCA1/2 mutant tumors.
Assuntos
Quebras de DNA de Cadeia Simples , DNA Primase/metabolismo , Replicação do DNA , DNA de Neoplasias/biossíntese , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Neoplasias/enzimologia , Nucleotidiltransferases/metabolismo , Reparo de DNA por Recombinação , Animais , Antineoplásicos/farmacologia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , DNA Primase/genética , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Feminino , Células HEK293 , Humanos , Camundongos Nus , Enzimas Multifuncionais/genética , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/genética , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
DNA Primase/metabolismo , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , DNA/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Primase/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Células HEK293 , Humanos , Enzimas Multifuncionais/fisiologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
The replisome must overcome DNA damage to ensure complete chromosome replication. Here, we describe the earliest events in this process by reconstituting collisions between a eukaryotic replisome, assembled with purified proteins, and DNA damage. Lagging-strand lesions are bypassed without delay, leaving daughter-strand gaps roughly the size of an Okazaki fragment. In contrast, leading-strand polymerase stalling significantly impacts replication fork progression. We reveal that the core replisome itself can bypass leading-strand damage by re-priming synthesis beyond it. Surprisingly, this restart activity is rare, mainly due to inefficient leading-strand re-priming, rather than single-stranded DNA exposure or primer extension. We find several unanticipated mechanistic distinctions between leading- and lagging-strand priming that we propose control the replisome's initial response to DNA damage. Notably, leading-strand restart was specifically stimulated by RPA depletion, which can occur under conditions of replication stress. Our results have implications for pathway choice at stalled forks and priming at DNA replication origins.
Assuntos
Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , DNA/metabolismo , Dano ao DNA/fisiologia , DNA Primase/metabolismo , Reparo do DNA/genética , DNA de Cadeia Simples/metabolismo , Eucariotos/genética , Células Eucarióticas/metabolismo , Origem de Replicação/genética , Origem de Replicação/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
G quadruplexes (G4s) can present potent blocks to DNA replication. Accurate and timely replication of G4s in vertebrates requires multiple specialized DNA helicases and polymerases to prevent genetic and epigenetic instability. Here we report that PrimPol, a recently described primase-polymerase (PrimPol), plays a crucial role in the bypass of leading strand G4 structures. While PrimPol is unable to directly replicate G4s, it can bind and reprime downstream of these structures. Disruption of either the catalytic activity or zinc-finger of PrimPol results in extreme G4-dependent epigenetic instability at the BU-1 locus in avian DT40 cells, indicative of extensive uncoupling of the replicative helicase and polymerase. Together, these observations implicate PrimPol in promoting restart of DNA synthesis downstream of, but closely coupled to, G4 replication impediments.
Assuntos
Proteínas Aviárias/metabolismo , DNA Primase/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Quadruplex G , Enzimas Multifuncionais/metabolismo , Animais , Proteínas Aviárias/genética , Sequência de Bases , Linhagem Celular , Galinhas , Montagem e Desmontagem da Cromatina , DNA/química , DNA Primase/genética , DNA Polimerase Dirigida por DNA/genética , Epigênese Genética , Instabilidade Genômica , Histonas/metabolismo , Dados de Sequência Molecular , Enzimas Multifuncionais/genética , TransfecçãoRESUMO
DNA replication forks are constantly challenged by DNA lesions induced by endogenous and exogenous sources. DNA damage tolerance mechanisms ensure that DNA replication continues with minimal effects on replication fork elongation either by using specialized DNA polymerases, which have the ability to replicate through the damaged template, or by skipping the damaged DNA, leaving it to be repaired after replication. These mechanisms are evolutionarily conserved in bacteria, yeast, and higher eukaryotes, and are paramount to ensure timely and faithful duplication of the genome. The Primase and DNA-directed Polymerase (PRIMPOL) is a recently discovered enzyme that possesses both primase and polymerase activities. PRIMPOL is emerging as a key player in DNA damage tolerance, particularly in vertebrate and human cells. Here, we review our current understanding of the function of PRIMPOL in DNA damage tolerance by focusing on the structural aspects that define its dual enzymatic activity, as well as on the mechanisms that control its chromatin recruitment and expression levels. We also focus on the latest findings on the mitochondrial and nuclear functions of PRIMPOL and on the impact of loss of these functions on genome stability and cell survival. Defining the function of PRIMPOL in DNA damage tolerance is becoming increasingly important in the context of human disease. In particular, we discuss recent evidence pointing at the PRIMPOL pathway as a novel molecular target to improve cancer cell response to DNA-damaging chemotherapy and as a predictive parameter to stratify patients in personalized cancer therapy.
Assuntos
Dano ao DNA/genética , DNA Primase/genética , DNA Primase/metabolismo , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular/genética , Cromatina/metabolismo , DNA/genética , DNA/metabolismo , DNA Primase/química , DNA Polimerase Dirigida por DNA/química , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Humanos , Mitocôndrias/metabolismo , Enzimas Multifuncionais/químicaRESUMO
During DNA replication, conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R-loops, three-stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single-stranded DNA, are important drivers of damage arising from such conflicts. How R-loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here, we show in vivo how R-loop formation drives a short purine-rich repeat, (GAA)10, to become a replication impediment that engages the repriming activity of the primase-polymerase PrimPol. Further, the absence of PrimPol leads to significantly increased R-loop formation around this repeat during S phase. We extend this observation by showing that PrimPol suppresses R-loop formation in genes harbouring secondary structure-forming sequences, exemplified by G quadruplex and H-DNA motifs, across the genome in both avian and human cells. Thus, R-loops promote the creation of replication blocks at susceptible structure-forming sequences, while PrimPol-dependent repriming limits the extent of unscheduled R-loop formation at these sequences, mitigating their impact on replication.
Assuntos
DNA Primase/metabolismo , Replicação do DNA , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/metabolismo , Quadruplex G , Enzimas Multifuncionais/metabolismo , Estruturas R-Loop , Fase S , Animais , Células Cultivadas , Galinhas , DNA Primase/genética , DNA de Cadeia Simples/química , DNA Polimerase Dirigida por DNA/genética , Drosophila , Humanos , Enzimas Multifuncionais/genéticaRESUMO
Background: New era of cardiac surgery aims to provide an enhanced postoperative recovery through the implementation of every step of the process. Thus, perfusion strategy should adopt evidence-based measures to reduce the impact of cardiopulmonary bypass (CPB). Hematic Antegrade Repriming (HAR) provides a standardized procedure combining several measures to reduce haemodilutional priming to 300 mL. Once the safety of the procedure in terms of embolic release has been proven, the evaluation of its beneficial effects in terms of transfusion and ICU stay should be assessed to determine if could be considered for inclusion in Enhanced Recovery After Cardiac Surgery (ERACS) programs. Methods: Two retrospective and non-randomized cohorts of high-risk patients, with similar characteristics, were assessed with a propensity score matching model. The treatment group (HG) (n = 225) received the HAR. A historical cohort, exposed to conventional priming with 1350 mL of crystalloid confirmed the control group (CG) (n = 210). Results: Exposure to any transfusion was lower in treated (66.75% vs. 6.88%, p < 0.01). Prolonged mechanical ventilation (>10 h) (26.51% vs. 12.62%; p < 0.01) and extended ICU stay (>2 d) (47.47% vs. 31.19%; p < 0.01) were fewer for treated. HAR did not increase early morbidity and mortality. Related savings varied from 581 to 2741.94 $/patient, depending on if direct or global expenses were considered. Discussion: By reducing the gaseous and crystalloid emboli during CPB initiation, HAR seems to have a beneficial impact on recovery and reduces the overall transfusion until discharge, leading to significant cost savings per process. Due to the preliminary and retrospective nature of the research and its limitations, our findings should be validated by future prospective and randomized studies.
Assuntos
Transfusão de Sangue , Ponte Cardiopulmonar , Recuperação Pós-Cirúrgica Melhorada , Humanos , Transfusão de Sangue/métodos , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Procedimentos Cirúrgicos Cardíacos/métodos , Ponte Cardiopulmonar/efeitos adversos , Ponte Cardiopulmonar/métodos , Estudos Retrospectivos , Resultado do TratamentoRESUMO
DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the Escherichia coli replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.
Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , DNA Bacteriano , DNA Polimerase Dirigida por DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
PrimPol, (primase-polymerase), the most recently identified eukaryotic polymerase, has roles in both nuclear and mitochondrial DNA maintenance. PrimPol is capable of acting as a DNA polymerase, with the ability to extend primers and also bypass a variety of oxidative and photolesions. In addition, PrimPol also functions as a primase, catalysing the preferential formation of DNA primers in a zinc finger-dependent manner. Although PrimPol's catalytic activities have been uncovered in vitro, we still know little about how and why it is targeted to the mitochondrion and what its key roles are in the maintenance of this multicopy DNA molecule. Unlike nuclear DNA, the mammalian mitochondrial genome is circular and the organelle has many unique proteins essential for its maintenance, presenting a differing environment within which PrimPol must function. Here, we discuss what is currently known about the mechanisms of DNA replication in the mitochondrion, the proteins that carry out these processes and how PrimPol is likely to be involved in assisting this vital cellular process.
Assuntos
DNA Primase/metabolismo , Replicação do DNA , DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Animais , Núcleo Celular/genética , DNA Primase/genética , DNA Polimerase Dirigida por DNA/genética , Humanos , Mitocôndrias/genética , Enzimas Multifuncionais/genética , MutaçãoRESUMO
Sodium channel Nav1.7 triggers the generation of nociceptive action potentials and is important in sending pain signals under physiological and pathological conditions. However, studying endogenous Nav1.7 currents has been confounded by co-expression of multiple sodium channel isoforms in dorsal root ganglion (DRG) neurons. In the current study, slow-repriming (SR) and fast-repriming (FR) tetrodotoxin-sensitive (TTX-S) currents were dissected electrophysiologically in small DRG neurons of both rats and mice. Three subgroups of small DRG neurons were identified based on the expression pattern of SR and FR TTX-S currents. A majority of rat neurons only expressed SR TTX-S currents, while a majority of mouse neurons expressed additional FR TTX-S currents. ProTx-II inhibited SR TTX-S currents with variable efficacy among DRG neurons. The expression of both types of TTX-S currents was higher in Isolectin B4-negative (IB4-) compared to Isolectin B4-positive (IB4+) neurons. Paclitaxel selectively increased SR TTX-S currents in IB4- neurons. In simulation experiments, the Nav1.7-expressing small DRG neuron displayed lower rheobase and higher frequency of action potentials upon threshold current injections compared to Nav1.6. The results suggested a successful dissection of endogenous Nav1.7 currents through electrophysiological manipulation that may provide a useful way to study the functional expression and pharmacology of endogenous Nav1.7 channels in DRG neurons.
RESUMO
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.
Assuntos
Enzimas Multifuncionais , Neoplasias , DNA Primase/química , DNA Primase/genética , DNA Primase/metabolismo , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , Humanos , Enzimas Multifuncionais/química , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
The eukaryotic replisome coordinates template unwinding and nascent-strand synthesis to drive DNA replication fork progression and complete efficient genome duplication. During its advancement along the parental template, each replisome may encounter an array of obstacles including damaged and structured DNA that impede its progression and threaten genome stability. A number of mechanisms exist to permit replisomes to overcome such obstacles, maintain their progression, and prevent fork collapse. A combination of recent advances in structural, biochemical, and single-molecule approaches have illuminated the architecture of the replisome during unperturbed replication, rationalised the impact of impediments to fork progression, and enhanced our understanding of DNA damage tolerance mechanisms and their regulation. This review focusses on these studies to provide an updated overview of the mechanisms that support replisomes to maintain their progression on an imperfect template.
RESUMO
Background: Chronic granulomatous disease (CGD) is caused by a malfunctioning nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in phagocytes, leading to impaired bacterial and fungal killing and hyperinflammation. Objective: To characterize macrophage subsets and cytokine/chemokine signaling loops involved in CGD tissue hyperinflammation. Methods: Cytokine/chemokine production and surface marker expression were analyzed in inflamed tissue of four CGD patients and compared to cytokine/chemokine released by CGD macrophages upon priming to different macrophage subpopulations. Furthermore, the re-priming capacity of CGD pro-inflammatory M1 to M2a anti-inflammatory macrophages was evaluated. Results: In human CGD inflammatory tissue, IL-18 and IFN-γ were detected in significant quantity. Immunofluorescence analysis identified macrophages as one source of IL-18 in inflamed tissue. In vitro, CGD macrophages could be primed and re-primed into all inflammatory/anti-inflammatory macrophage subpopulations. IL-18 was also released by M1 CGD and control macrophages. Conclusion: CGD pro-inflammatory M1 macrophages remain M1 primed in vivo. As CGD M1 macrophages can be re-primed to anti-inflammatory M2a phenotype in vitro, macrophages are kept in M1 state in vivo by a persistent pro-inflammatory environment. Our results suggest a paracrine signaling loop between M1 macrophage derived IL-18 and non-macrophage derived IFN-γ maintaining macrophage pro-inflammatory activity in CGD tissue.
Assuntos
Doença Granulomatosa Crônica/imunologia , Interferon gama/imunologia , Interleucina-18/imunologia , Macrófagos/imunologia , Comunicação Parácrina/imunologia , Transdução de Sinais/imunologia , Adolescente , Adulto , Criança , Feminino , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/patologia , Humanos , Lactente , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interferon gama/genética , Interleucina-18/genética , Macrófagos/patologia , Masculino , Comunicação Parácrina/genética , Transdução de Sinais/genéticaRESUMO
Genome duplication is coupled with DNA damage tolerance (DDT) and chromatin structural changes. Recently we reported that mutations in Primase subunits or factors that bridge Polα/Primase with the replicative helicase, Ctf4, caused abnormal usage of DDT pathways, negatively influenced sister chromatid cohesion (SCC), and associated with increased fork reversal. (1) We also found that cohesin, which is paradigmatic for SCC, facilitates recombination-mediated DDT. However, only the recombination defects of cohesin, but not of cohesion-defective Polα/Primase/Ctf4 mutants, were rescued by artificial tethering of sister chromatids. Genetic tests and electron microscopy analysis of replication intermediates made us propose that management of single-stranded DNA forming proximal to the fork is a critical determinant of chromosome and replication fork structure, and influences DDT pathway choice. Here we discuss the implications of our findings for understanding DDT regulation and cohesion establishment during replication, and outline directions to rationalize the relationship between these chromosome metabolism processes.
Assuntos
DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Replicação do DNA/fisiologia , DNA Fúngico/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Polimerase I/genética , DNA Primase/genética , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
PrimPol is a DNA damage tolerance enzyme possessing both translesion synthesis (TLS) and primase activities. To uncover its potential role in TLS-mediated IgVλ hypermutation and define its interplay with other TLS polymerases, PrimPol(-/-) and PrimPol(-/-)/Polη(-/-)/Polζ (-/-) gene knockouts were generated in avian cells. Loss of PrimPol had no significant impact on the rate of hypermutation or the mutation spectrum of IgVλ. However, PrimPol(-/-) cells were sensitive to methylmethane sulfonate, suggesting that it may bypass abasic sites at the IgVλ segment by repriming DNA synthesis downstream of these sites. PrimPol(-/-) cells were also sensitive to cisplatin and hydroxyurea, indicating that it assists in maintaining / restarting replication at a variety of lesions. To accurately measure the relative contribution of the TLS and primase activities, we examined DNA damage sensitivity in PrimPol(-/-) cells complemented with polymerase or primase-deficient PrimPol. Polymerase-defective, but not primase-deficient, PrimPol suppresses the hypersensitivity of PrimPol(-/-) cells. This indicates that its primase, rather than TLS activity, is pivotal for DNA damage tolerance. Loss of TLS polymerases, Polη and Polζ has an additive effect on the sensitivity of PrimPol(-/-) cells. Moreover, we found that PrimPol and Polη-Polζ redundantly prevented cell death and facilitated unperturbed cell cycle progression. PrimPol(-/-) cells also exhibited increased sensitivity to a wide variety of chain-terminating nucleoside analogs (CTNAs). PrimPol could perform close-coupled repriming downstream of CTNAs and oxidative damage in vitro. Together, these results indicate that PrimPol's repriming activity plays a central role in reinitiating replication downstream from CTNAs and other specific DNA lesions.
Assuntos
Dano ao DNA , DNA Primase/metabolismo , Replicação do DNA , Nucleosídeos/metabolismo , Animais , Biocatálise/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Galinhas , Cisplatino/farmacologia , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , DNA Polimerase Dirigida por DNA/metabolismo , Região Variável de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/genética , Metanossulfonato de Metila/farmacologia , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Raios UltravioletaRESUMO
Genome duplication is temporarily coordinated with sister chromatid cohesion and DNA damage tolerance. Recently, we found that replication fork-coupled repriming is important for both optimal cohesion and error-free replication by recombination. The mechanism involved has implications for the etiology of replication-based genetic diseases and cancer.
RESUMO
The DNA-directed primase-polymerase PrimPol of the archaeo-eukaryotic primase superfamily represents an ancient solution to the many problems faced during genome duplication. This versatile enzyme is capable of initiating de novo DNA/RNA synthesis, DNA chain elongation, and has the capacity to bypass modifications that stall the replisome by trans-lesion synthesis or origin-independent re-priming, thus allowing discontinuous synthesis of the leading strand. Recent studies have shown that PrimPol is an important new player in replication fork progression in eukaryotic cells; this review summarizes our current understanding of PrimPol and highlights important questions that remain to be addressed.