RESUMO
Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of the same molecule cooperate for substrates binding and catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5'-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that the NTD alone is able to initiate DNA synthesis, and the CTD stimulates the primase activity of NTD. The regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA is also demonstrated.
Assuntos
DNA Primase , DNA Polimerase Dirigida por DNA , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , DNA Primase/metabolismo , Replicação do DNA , DNA/genética , Primers do DNA , Catálise , Enzimas Multifuncionais/químicaRESUMO
Transmission of genetic information depends on successful completion of DNA replication. Genomic DNA is subjected to damage on a daily basis. DNA lesions create obstacles for DNA polymerases and can lead to the replication blockage, formation of DNA breaks, cell cycle arrest, and apoptosis. Cells have evolutionary adapted to DNA damage by developing mechanisms allowing elimination of lesions prior to DNA replication (DNA repair) and helping to bypass lesions during DNA synthesis (DNA damage tolerance). The second group of mechanisms includes the restart of DNA synthesis at the sites of DNA damage by DNA primase-polymerase PrimPol. Human PrimPol was described in 2013. The properties and functions of this enzyme have been extensively studied in recent years, but very little is known about the regulation of PrimPol and association between the enzyme dysfunction and diseases. In this review, we described the mechanisms of human PrimPol regulation in the context of DNA replication, discussed in detail interactions of PrimPol with other proteins, and proposed possible pathways for the regulation of human PrimPol activity. The article also addresses the association of PrimPol dysfunction with human diseases.
Assuntos
DNA Primase , DNA Polimerase Dirigida por DNA , Humanos , DNA Primase/genética , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Replicação do DNA , DNA/metabolismo , Dano ao DNA , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismoRESUMO
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.
Assuntos
Adenocarcinoma de Pulmão , DNA Polimerase Dirigida por DNA , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , Células A549 , Cisplatino/farmacologia , Peróxido de Hidrogênio/farmacologia , Replicação do DNA , Dano ao DNA , Adenocarcinoma de Pulmão/genética , DNA Primase/genética , DNA Primase/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismoRESUMO
DNA-protein cross-links remain the least-studied type of DNA damage. Recently, their repair was shown to involve proteolysis; however, the fate of the peptide remnant attached to DNA is unclear. Particularly, peptide cross-links could interfere with DNA polymerases. Apurinuic/apyrimidinic (AP) sites, abundant and spontaneously arising DNA lesions, readily form cross-links with proteins. Their degradation products (AP site-peptide cross-links, APPXLs) are non-instructive and should be even more problematic for polymerases. Here, we address the ability of human DNA polymerases involved in DNA repair and translesion synthesis (POLß, POLλ, POLη, POLκ and PrimPOL) to carry out synthesis on templates containing AP sites cross-linked to the N-terminus of a 10-mer peptide (APPXL-I) or to an internal lysine of a 23-mer peptide (APPXL-Y). Generally, APPXLs strongly blocked processive DNA synthesis. The blocking properties of APPXL-I were comparable with those of an AP site, while APPXL-Y constituted a much stronger obstruction. POLη and POLκ demonstrated the highest bypass ability. DNA polymerases mostly used dNTP-stabilized template misalignment to incorporate nucleotides when encountering an APPXL. We conclude that APPXLs are likely highly cytotoxic and mutagenic intermediates of AP site-protein cross-link repair and must be quickly eliminated before replication.
Assuntos
Reparo do DNA , DNA Polimerase Dirigida por DNA , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Replicação do DNA , Dano ao DNA , Nucleotídeos , PeptídeosRESUMO
Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and possesses the conditional strand displacement activity stimulated by Mn2+ ions and accessory replicative proteins RPA and PolDIP2. The DNA displacement activity of PrimPol was found to be more efficient than the RNA displacement activity and FEN1 processed the 5'-DNA flaps generated by PrimPol in vitro.
Assuntos
DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Endonucleases Flap/metabolismo , Humanos , Manganês/farmacologia , Proteínas Nucleares/metabolismo , RNA/metabolismo , Proteína de Replicação A/metabolismo , Especificidade por Substrato/efeitos dos fármacosRESUMO
Transcripts of many enzymes involved in base excision repair (BER) undergo extensive alternative splicing, but functions of the corresponding alternative splice variants remain largely unexplored. In this review, we cover the studies describing the common alternatively spliced isoforms and disease-associated variants of DNA glycosylases, AP-endonuclease 1, and DNA polymerase beta. We also discuss the roles of alternative splicing in the regulation of their expression, catalytic activities, and intracellular transport.
Assuntos
Processamento Alternativo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Isoformas de Proteínas , Animais , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Enzimas Reparadoras do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Humanos , Camundongos , RNA Mensageiro/metabolismo , RatosRESUMO
PrimPol is a human deoxyribonucleic acid (DNA) polymerase that also possesses primase activity and is involved in DNA damage tolerance, the prevention of genome instability and mitochondrial DNA maintenance. In this review, we focus on recent advances in biochemical and crystallographic studies of PrimPol, as well as in identification of new protein-protein interaction partners. Furthermore, we discuss the possible functions of PrimPol in both the nucleus and the mitochondria.
Assuntos
Dano ao DNA , DNA Primase/química , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/química , Enzimas Multifuncionais/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cristalografia , Replicação do DNA , DNA Mitocondrial/genética , Instabilidade Genômica , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismoRESUMO
PrimPol is a human DNA primase involved in DNA damage tolerance pathways by restarting DNA replication downstream of DNA lesions and non-canonical DNA structures. Activity and affinity to DNA relays on the interaction of PrimPol with replication protein A (RPA). In this work, we report that PrimPol has an intrinsic ability to copy DNA hairpins with a stem length of 5-9 base pairs (bp) but shows pronounced pausing of DNA synthesis. RPA greatly stimulates DNA synthesis across inverted DNA repeats by PrimPol. Moreover, deletion of the C-terminal RPA binding motif (RBM) facilitates DNA hairpin bypass and makes it independent of RPA. This work supports the idea that RBM is a negative regulator of PrimPol and its interaction with RPA is required to achieve the fully active state.
RESUMO
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.
Assuntos
DNA Primase , DNA Polimerase Dirigida por DNA , Enzimas Multifuncionais , Mutação de Sentido Incorreto , Neoplasias Ovarianas , Neoplasias do Colo do Útero , Feminino , Humanos , Substituição de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , DNA Primase/metabolismo , DNA Primase/química , DNA Primase/genética , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/química , Modelos Moleculares , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/química , Conformação Proteica , Neoplasias do Colo do Útero/genética , Neoplasias Ovarianas/genéticaRESUMO
Epigenetic cytosine methylation covers most of genomic CpG dinucleotides in human cells. In addition to common deamination-mediated mutagenesis at CpG sites, an alternative deamination-independent pathway associated with DNA polymerase activity was previously described. This mutagenesis is characterized by the TCGâTTG mutational signature and is believed to arise from dAMP misincorporation opposite 5-methylcytosine (mC) or its oxidized derivative 5-hydroxymethylcytosine (hmC) by B-family replicative DNA polymerases with disrupted proofreading 3â5'-exonuclease activity. In addition to being less stable and pro-mutagenic themselves, cytosine modifications also increase the risk of adjacent nucleotides damage, including the formation of 8-oxo-2'-deoxyguanosine (8-oxoG), a well-known mutagenic lesion. The effect of cytosine methylation on error-prone DNA polymerases lacking proofreading activity and involved in repair and DNA translesion synthesis remains unexplored. Here we analyze the efficiency and fidelity of translesion Y-family polymerases (Pol κ, Pol η, Pol ι and REV1) and primase-polymerase PrimPol opposite mC and hmC as well as opposite 8-oxoG adjacent to mC in the TCG context. We demonstrate that epigenetic cytosine modifications suppress Pol ι and REV1 activities and lead to increasing dAMP misincorporation by PrimPol, Pol κ and Pol ι in vitro. Cytosine methylation also increases misincorporation of dAMP opposite the adjacent 8-oxoG by PrimPol, decreases the TLS activity of Pol η opposite the lesion but increases dCMP incorporation opposite 8-oxoG by REV1. Altogether, these data suggest that methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.
Assuntos
5-Metilcitosina , Citosina , Metilação de DNA , DNA Polimerase Dirigida por DNA , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , Citosina/metabolismo , Citosina/análogos & derivados , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , Reparo do DNA , Dano ao DNA , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , DNA Polimerase iota , DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Replicação do DNA , 8-Hidroxi-2'-Desoxiguanosina/metabolismoRESUMO
Primase-DNA polymerase (PrimPol) is involved in reinitiating DNA synthesis at stalled replication forks. PrimPol also possesses DNA translesion (TLS) activity and bypasses several endogenous nonbulky DNA lesions in vitro. Little is known about the TLS activity of PrimPol across bulky carcinogenic adducts. We analyzed the DNA polymerase activity of human PrimPol on DNA templates with seven N2-dG lesions of different steric bulkiness. In the presence of Mg2+ ions, bulky N2-isobutyl-dG, N2-benzyl-dG, N2-methyl(1-naphthyl)-dG, N2-methyl(9-anthracenyl)-dG, N2-methyl(1-pyrenyl)-dG, and N2-methyl(1,3-dimethoxyanthraquinone)-dG adducts fully blocked PrimPol activity. At the same time, PrimPol incorporated complementary deoxycytidine monophosphate (dCMP) opposite N2-ethyl-dG with moderate efficiency but did not extend DNA beyond the lesion. We also demonstrated that mutation of the Arg288 residue abrogated dCMP incorporation opposite the lesion in the presence of Mn2+ ions. When Mn2+ replaced Mg2+, PrimPol carried out DNA synthesis on all DNA templates with N2-dG adducts in standing start reactions with low efficiency and accuracy, possibly utilizing a lesion "skipping" mechanism. The TLS activity of PrimPol opposite N2-ethyl-dG but not bulkier adducts was stimulated by accessory proteins, polymerase delta-interacting protein 2 (PolDIP2), and replication protein A (RPA). Molecular dynamics studies demonstrated the absence of stable interactions with deoxycytidine triphosphate (dCTP), large reactions, and C1'-C1' distances for the N2-isobutyl-dG and N2-benzyl-dG PrimPol complexes, suggesting that the size of the adduct is a limiting factor for efficient TLS across minor groove adducts by PrimPol.
Assuntos
Dano ao DNA , Desoxicitidina Monofosfato , Humanos , Desoxiguanosina/química , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/química , Adutos de DNA , Proteínas Nucleares/metabolismo , DNA Primase/metabolismo , Enzimas Multifuncionais/metabolismoRESUMO
Human primase and DNA polymerase PrimPol re-starts stalled replication forks by repriming downstream DNA lesions and protects cells against DNA damage. Structure of the catalytic core of PrimPol with DNA primer, template and incoming dATP was solved but the mechanisms of DNA polymerase and primase activities of PrimPol are not fully understood. In this work, using site-directed mutagenesis we biochemically analyzed the role of active site residues Arg47 and Arg76 contacting DNA template in DNA polymerase and primase activities of PrimPol. The substitution R47A diminished the DNA polymerase and primase activities of PrimPol whereas the single amino acid substitution R76A caused almost complete loss of catalytic activities. Both amino acid substitutions affected the spectrum of dNMPs incorporation on undamaged DNA templates and opposite 8-oxoguanine. Finally, substitutions of the Arg47 and Arg76 residues attenuated the formation of the stable PrimPol:DNA complex in the presence of ATP/dNTPs. Together, these findings suggest a key role of the Arg47 and Arg76 in DNA synthesis by PrimPol.
Assuntos
Domínio Catalítico , Dano ao DNA , DNA Primase/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Guanina/análogos & derivados , Enzimas Multifuncionais/metabolismo , Sequência de Aminoácidos , DNA Primase/química , DNA Polimerase Dirigida por DNA/química , Guanina/metabolismo , Humanos , Enzimas Multifuncionais/química , Alinhamento de SequênciaRESUMO
Human PrimPol belongs to the archaeo-eukaryotic primase superfamily of primases and is involved in de novo DNA synthesis downstream of blocking DNA lesions and non-B DNA structures. PrimPol possesses both DNA/RNA primase and DNA polymerase activities, and also bypasses a number of DNA lesions in vitro. In this work, we have analyzed translesion synthesis activity of PrimPol in vitro on DNA with an 1,2-intrastrand cisplatin cross-link (1,2-GG CisPt CL) or a model DNA-protein cross-link (DpCL). PrimPol was capable of the 1,2-GG CisPt CL bypass in the presence of Mn2+ ions and preferentially incorporated two complementary dCMPs opposite the lesion. Nucleotide incorporation was stimulated by PolDIP2, and yeast Pol ζ efficiently extended from the nucleotides inserted opposite the 1,2-GG CisPt CL in vitro. DpCLs significantly blocked the DNA polymerase activity and strand displacement synthesis of PrimPol. However, PrimPol was able to reach the DpCL site in single strand template DNA in the presence of both Mg2+ and Mn2+ ions despite the presence of the bulky protein obstacle.
Assuntos
Cisplatino/química , Dano ao DNA , DNA Primase/química , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA/química , Enzimas Multifuncionais/química , Cisplatino/farmacologia , Reagentes de Ligações Cruzadas/química , Reparo do DNA , HumanosRESUMO
Eukaryotic DNA polymerase eta (Pol η) plays a key role in the efficient and accurate DNA translesion synthesis (TLS) opposite UV-induced thymine dimers. Pol η is also involved in bypass of many other DNA lesions but possesses low fidelity on undamaged DNA templates. To better understand the mechanism of DNA synthesis by Pol η we investigated substitutions of evolutionary conserved active site residues Gln55 and Arg73 in Saccharomyces cerevisiae Pol η. We analyzed the efficiency and fidelity of DNA synthesis by the mutant Pol η variants opposite thymine dimers, abasic site, thymine glycol, 8-oxoguanine and on undamaged DNA. Substitutions Q55A and R73A decreased the catalytic activity and significantly affected DNA damage bypass by Pol η. In particular, the Q55A substitution reduced the efficiency of thymine dimers bypass, R73A had a stronger effect on the TLS-activity opposite abasic site, while both substitutions impaired replication opposite thymine glycol. Importantly, the R73A substitution also increased the fidelity of Pol η. Altogether, these results reveal a key role of residues Gln55 and Arg73 in DNA synthesis opposite various types of DNA lesions and highlight the evolutionary importance of the Pol η TLS function at the cost of DNA replication accuracy.
Assuntos
Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Arginina/genética , Arginina/metabolismo , Domínio Catalítico , Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Glicina/química , Glicina/genética , Glicina/metabolismo , Guanina/análogos & derivados , Guanina/química , Mutagênese Sítio-Dirigida , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Timina/análogos & derivados , Timina/químicaRESUMO
Many human DNA polymerases bypass DNA damage during translesion synthesis (TLS). Human primase and polymerase, PrimPol, assists fork progression by repriming DNA synthesis downstream of the lesion using its DNA primase activity. We tested the properties of PrimPol as a TLS polymerase in the presence of different metal ions in vitro. We demonstrate that in the presence of Mg2+ ions PrimPol carries out efficient and relatively accurate synthesis past 8-oxoguanine and 5-formyluracil. It also bypasses an abasic site and O6-methylguanine, but is blocked by thymine glycol and 1,N6-ethenoadenine. Substitution of Mg2+ with Mn2+ stimulates the TLS activity of PrimPol and allows for efficient, but error-prone, synthesis on DNA templates containing all tested DNA lesions, including thymine glycol and 1,N6-ethenoadenine. The TLS activity of PrimPol has possible relevant functions in vivo; e.g., the combined primase and DNA polymerase activities of PrimPol might facilitate replication of DNA with clustered damage.
Assuntos
DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Adenina/análogos & derivados , Adenina/metabolismo , Dano ao DNA , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Magnésio/farmacologia , Uracila/análogos & derivados , Uracila/metabolismoRESUMO
Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6-6.5, low salt concentrations and 3 mM Mg2+ or 0.3-3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37ºC. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol.