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1.
PLoS Genet ; 16(5): e1008755, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32379761

RESUMO

During eukaryotic DNA replication, DNA polymerase alpha/primase (Pol α) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol α associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol α abundance, and that both processes are disrupted at similar Pol α concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol α, lagging-strand initiation is impaired at Pol α concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol α. Using strains in which the Pol α-Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol α recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol α at the replication fork, at least when Pol α is limiting.


Assuntos
DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Replicação do DNA , DNA Fúngico/biossíntese , Proteínas de Ligação a DNA/fisiologia , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/fisiologia , DNA , Replicação do DNA/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Organismos Geneticamente Modificados , Ligação Proteica , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Elongação da Transcrição Genética/fisiologia
2.
Nucleic Acids Res ; 48(7): 3657-3677, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32128579

RESUMO

DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (KD values of 10-30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.


Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteína de Replicação A/metabolismo , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , DNA de Cadeia Simples/química , Ligação Proteica , Vírus 40 dos Símios/imunologia
3.
Cancer Res ; 80(8): 1735-1747, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32161100

RESUMO

Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (POLA1, POLE, and POLE2) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies.


Assuntos
Afidicolina/farmacologia , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Retinoides/farmacologia , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Dano ao DNA , DNA Polimerase I/antagonistas & inibidores , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , DNA Polimerase II/antagonistas & inibidores , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Polimerase beta , Drogas em Investigação/farmacologia , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Interferente Pequeno/análise , RNA Interferente Pequeno/genética
4.
Elife ; 82019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31589141

RESUMO

The current view is that eukaryotic replisomes are independent. Here we show that Ctf4 tightly dimerizes CMG helicase, with an extensive interface involving Psf2, Cdc45, and Sld5. Interestingly, Ctf4 binds only one Pol α-primase. Thus, Ctf4 may have evolved as a trimer to organize two helicases and one Pol α-primase into a replication factory. In the 2CMG-Ctf43-1Pol α-primase factory model, the two CMGs nearly face each other, placing the two lagging strands toward the center and two leading strands out the sides. The single Pol α-primase is centrally located and may prime both sister replisomes. The Ctf4-coupled-sister replisome model is consistent with cellular microscopy studies revealing two sister forks of an origin remain attached and are pushed forward from a protein platform. The replication factory model may facilitate parental nucleosome transfer during replication.


Assuntos
DNA Polimerase I/metabolismo , Replicação do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Ligação Proteica , Multimerização Proteica
5.
Mol Cell ; 76(3): 371-381.e4, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31495565

RESUMO

Break-induced replication (BIR) is a pathway of homology-directed repair that repairs one-ended DNA breaks, such as those formed at broken replication forks or uncapped telomeres. In contrast to conventional S phase DNA synthesis, BIR proceeds by a migrating D-loop and results in conservative synthesis of the nascent strands. DNA polymerase delta (Pol δ) initiates BIR; however, it is not known whether synthesis of the invading strand switches to a different polymerase or how the complementary strand is synthesized. By using alleles of the replicative DNA polymerases that are permissive for ribonucleotide incorporation, thus generating a signature of their action in the genome that can be identified by hydrolytic end sequencing, we show that Pol δ replicates both the invading and the complementary strand during BIR. In support of this conclusion, we show that depletion of Pol δ from cells reduces BIR, whereas depletion of Pol ε has no effect.


Assuntos
Quebras de DNA , DNA Polimerase III/metabolismo , Replicação do DNA , DNA Fúngico/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Polimerase III/genética , DNA Fúngico/genética , Células HEK293 , Células HeLa , Humanos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
6.
Mitochondrion ; 49: 166-177, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31445096

RESUMO

Human and yeast mitochondrial DNA polymerases (DNAPs), POLG and Mip1, are related by evolution to bacteriophage DNAPs. However, mitochondrial DNAPs contain unique amino and carboxyl-terminal extensions that physically interact. Here we describe that N-terminal deletions in Mip1 polymerases abolish polymerization and decrease exonucleolytic degradation, whereas moderate C-terminal deletions reduce polymerization. Similarly, to the N-terminal deletions, an extended C-terminal deletion of 298 amino acids is deficient in nucleotide addition and exonucleolytic degradation of double and single-stranded DNA. The latter observation suggests that the physical interaction between the amino and carboxyl-terminal regions of Mip1 may be related to the spread of pathogenic POLG mutant along its primary sequence.


Assuntos
DNA Polimerase I/metabolismo , DNA Fúngico/biossíntese , DNA Mitocondrial/biossíntese , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Domínio Catalítico , DNA Polimerase I/genética , Polimerase do DNA Mitocondrial/genética , Polimerase do DNA Mitocondrial/metabolismo , DNA Fúngico/genética , DNA Mitocondrial/genética , Humanos , Proteínas Mitocondriais/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
7.
Nucleic Acids Res ; 47(16): 8521-8536, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31251806

RESUMO

DNA replication forks are intrinsically asymmetric and may arrest during the cell cycle upon encountering modifications in the DNA. We have studied real time dynamics of three DNA polymerases and an exonuclease at a single molecule level in the bacterium Bacillus subtilis. PolC and DnaE work in a symmetric manner and show similar dwell times. After addition of DNA damage, their static fractions and dwell times decreased, in agreement with increased re-establishment of replication forks. Only a minor fraction of replication forks showed a loss of active polymerases, indicating relatively robust activity during DNA repair. Conversely, PolA, homolog of polymerase I and exonuclease ExoR were rarely present at forks during unperturbed replication but were recruited to replications forks after induction of DNA damage. Protein dynamics of PolA or ExoR were altered in the absence of each other during exponential growth and during DNA repair, indicating overlapping functions. Purified ExoR displayed exonuclease activity and preferentially bound to DNA having 5' overhangs in vitro. Our analyses support the idea that two replicative DNA polymerases work together at the lagging strand whilst only PolC acts at the leading strand, and that PolA and ExoR perform inducible functions at replication forks during DNA repair.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , DNA Polimerase I/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Regulação Bacteriana da Expressão Gênica , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Dano ao DNA , DNA Polimerase I/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Replicação do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
Nucleic Acids Res ; 47(13): 6973-6983, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31170294

RESUMO

Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2'-deoxy-2'-fluoro-ß-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.


Assuntos
Proteínas de Bactérias/química , DNA Polimerase I/química , Geobacillus stearothermophilus/enzimologia , DNA Polimerase Dirigida por RNA/química , Arabinonucleotídeos/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , DNA Polimerase I/isolamento & purificação , DNA Polimerase I/metabolismo , DNA Bacteriano/metabolismo , Modelos Moleculares , Hibridização de Ácido Nucleico , Nucleosídeos/metabolismo , Ligação Proteica , Conformação Proteica , DNA Polimerase Dirigida por RNA/isolamento & purificação , DNA Polimerase Dirigida por RNA/metabolismo , Relação Estrutura-Atividade , Moldes Genéticos
9.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30979824

RESUMO

Human CTC1-STN1-TEN1 (CST) is an RPA-like single-stranded DNA-binding protein that interacts with DNA polymerase α-primase (pol α) and functions in telomere replication. Previous studies suggest that CST also promotes replication restart after fork stalling. However, the precise role of CST in genome-wide replication remains unclear. In this study, we sought to understand whether CST alters origin licensing and activation. Replication origins are licensed by loading of the minichromosome maintenance 2-7 (MCM) complex in G1 followed by replisome assembly and origin firing in S-phase. We find that CST directly interacts with the MCM complex and disrupts binding of CDT1 to MCM, leading to decreased origin licensing. We also show that CST enhances replisome assembly by promoting AND-1/pol α chromatin association. Moreover, these interactions are not dependent on exogenous replication stress, suggesting that CST acts as a specialized replication factor during normal replication. Overall, our findings implicate CST as a novel regulator of origin licensing and replisome assembly/fork progression through interactions with MCM, AND-1, and pol α.


Assuntos
Cromatina/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA Polimerase I/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Técnicas de Silenciamento de Genes , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Proteínas de Manutenção de Minicromossomo/metabolismo , RNA Interferente Pequeno/genética , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética
10.
PLoS Pathog ; 15(4): e1007742, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31026293

RESUMO

Persistent hepatitis B virus (HBV) infection relies on the establishment and maintenance of covalently closed circular (ccc) DNA, a 3.2 kb episome that serves as a viral transcription template, in the nucleus of an infected hepatocyte. Although evidence suggests that cccDNA is the repair product of nucleocapsid associated relaxed circular (rc) DNA, the cellular DNA polymerases involving in repairing the discontinuity in both strands of rcDNA as well as the underlying mechanism remain to be fully understood. Taking a chemical genetics approach, we found that DNA polymerase alpha (Pol α) is essential for cccDNA intracellular amplification, a genome recycling pathway that maintains a stable cccDNA pool in infected hepatocytes. Specifically, inhibition of Pol α by small molecule inhibitors aphidicolin or CD437 as well as silencing of Pol α expression by siRNA led to suppression of cccDNA amplification in human hepatoma cells. CRISPR-Cas9 knock-in of a CD437-resistant mutation into Pol α genes completely abolished the effect of CD437 on cccDNA formation, indicating that CD437 directly targets Pol α to disrupt cccDNA biosynthesis. Mechanistically, Pol α is recruited to HBV rcDNA and required for the generation of minus strand covalently closed circular rcDNA, suggesting that Pol α is involved in the repair of the minus strand DNA nick in cccDNA synthesis. Our study thus reveals that the distinct host DNA polymerases are hijacked by HBV to support the biosynthesis of cccDNA from intracellular amplification pathway compared to that from de novo viral infection, which requires Pol κ and Pol λ.


Assuntos
DNA Polimerase I/metabolismo , DNA Circular/genética , DNA Viral/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Replicação Viral/genética , DNA Circular/metabolismo , DNA Viral/metabolismo , Células Hep G2 , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/metabolismo , Hepatite B Crônica/virologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Vírion
11.
Nucleic Acids Res ; 47(10): 5243-5259, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30976812

RESUMO

Human CST (CTC1-STN1-TEN1) is an RPA-like complex that associates with G-rich single-strand DNA and helps resolve replication problems both at telomeres and genome-wide. We previously showed that CST binds and disrupts G-quadruplex (G4) DNA in vitro, suggesting that CST may prevent in vivo blocks to replication by resolving G4 structures. Here, we demonstrate that CST binds and unfolds G4 with similar efficiency to RPA. In cells, CST is recruited to telomeric and non-telomeric chromatin upon G4 stabilization, even when ATR/ATM pathways were inhibited. STN1 depletion increases G4 accumulation and slows bulk genomic DNA replication. At telomeres, combined STN1 depletion and G4 stabilization causes multi-telomere FISH signals and telomere loss, hallmarks of deficient telomere duplex replication. Strand-specific telomere FISH indicates preferential loss of C-strand DNA while analysis of BrdU uptake during leading and lagging-strand telomere replication shows preferential under-replication of lagging telomeres. Together these results indicate a block to Okazaki fragment synthesis. Overall, our findings indicate a novel role for CST in maintaining genome integrity through resolution of G4 structures both ahead of the replication fork and on the lagging strand template.


Assuntos
Quadruplex G , Proteínas de Ligação a Telômeros/genética , Animais , Linhagem Celular , DNA , DNA Polimerase I/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Insetos , Cinética , Ligação Proteica , Telomerase/metabolismo , Telômero/metabolismo
12.
PLoS One ; 14(4): e0215411, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30970012

RESUMO

We report here on the stability and catalytic properties of the HoLaMa DNA polymerase, a Klenow sub-fragment lacking the 3'-5' exonuclease domain. HoLaMa was overexpressed in Escherichia coli, and the enzyme was purified by means of standard chromatographic techniques. High-resolution NMR experiments revealed that HoLaMa is properly folded at pH 8.0 and 20°C. In addition, urea induced a cooperative folding to unfolding transition of HoLaMa, possessing an overall thermodynamic stability and a transition midpoint featuring ΔG and CM equal to (15.7 ± 1.9) kJ/mol and (3.5 ± 0.6) M, respectively. When the catalytic performances of HoLaMa were compared to those featured by the Klenow enzyme, we did observe a 10-fold lower catalytic efficiency by the HoLaMa enzyme. Surprisingly, HoLaMa and Klenow DNA polymerases possess markedly different sensitivities in competitive inhibition assays performed to test the effect of single dNTPs.


Assuntos
DNA Polimerase I/química , DNA Polimerase I/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Substituição de Aminoácidos , Domínio Catalítico , DNA Polimerase I/genética , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Cinética , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios Proteicos , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica
13.
Nucleic Acids Res ; 47(7): 3422-3433, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30759238

RESUMO

The developmental asymmetry of fission yeast daughter cells derives from inheriting 'older Watson' versus 'older Crick' DNA strand from the parental cell, strands that are complementary but not identical with each other. A novel DNA strand-specific 'imprint', installed during DNA replication at the mating-type locus (mat1), imparts competence for cell type inter-conversion to one of the two chromosome replicas. The catalytic subunit of DNA Polymerase α (Polα) has been implicated in the imprinting process. Based on its known biochemical function, Polα might install the mat1 imprint during lagging strand synthesis. The nature of the imprint is not clear: it is either a nick or a ribonucleotide insertion. Our investigations do not support a direct role of Polα in nicking through putative endonuclease domains but confirm its indirect role in installing an alkali-labile moiety as the imprint. While ruling out the role of the primase subunit of Polα holoenzyme, we find that mutations in the Polα-recruitment and putative primase homology domain in Mcm10/Cdc23 abrogate the ribonucleotide imprint formation. These results, while confirming the ribonucleotide nature of the imprint suggest the possibility of a direct role of Mcm10/Cdc23 in installing it in cooperation with Polα and Swi1.


Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Polimerase I/metabolismo , Replicação do DNA/genética , Genes Fúngicos Tipo Acasalamento/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Ribonucleotídeos/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Domínio Catalítico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , DNA Polimerase I/química , DNA Polimerase I/genética , DNA Primase/química , DNA Primase/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética
14.
PLoS One ; 14(2): e0211653, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30726292

RESUMO

DNA deamination generates base transitions and apurinic/apyrimidinic (AP)-sites which are potentially genotoxic and cytotoxic. In Bacillus subtilis uracil can be removed from DNA by the uracil DNA-glycosylase through the base excision repair pathway. Genetic evidence suggests that B. subtilis YwqL, a homolog of Endonuclease-V (EndoV), acts on a wider spectrum of deaminated bases but the factors that complete this pathway have remained elusive. Here, we report that a purified His6-YwqL (hereafter BsEndoV) protein had in vitro endonuclease activity against double-stranded DNAs containing a single uracil (U), hypoxanthine (Hx), xanthine (X) or an AP site. Interestingly, while BsEndoV catalyzed a single strand break at the second phosphodiester bond towards the 3'-end of the U and AP lesions, there was an additional cleavage of the phosphodiester bond preceding the Hx and X lesions. Remarkably, the repair event initiated by BsEndoV on Hx and X, was completed by a recombinant B. subtilis His6-DNA polymerase A (BsPolA), but not on BsEndoV-processed U and AP lesions. For the latter lesions a second excision event performed by a recombinant B. subtilis His6-ExoA (BsExoA) was necessary before completion of their repair by BsPolA. These results suggest the existence of a novel alternative excision repair pathway in B. subtilis that counteracts the genotoxic effects of base deamination. The presence of this novel pathway in vivo in B. subtilis was also supported by analysis of effects of single or multiple deletions of exoA, endoV and polA on spontaneous mutations in growing cells, and the sensitivity of growing wild-type and mutant cells to a DNA deaminating agent.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , DNA Polimerase I/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , DNA Polimerase I/genética , Desaminação , Desoxirribonuclease (Dímero de Pirimidina)/genética , Mutagênese , Proteínas Recombinantes
15.
J Bacteriol ; 201(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30670546

RESUMO

RNA-DNA hybrids are common in chromosomal DNA. Persistent RNA-DNA hybrids result in replication fork stress, DNA breaks, and neurological disorders in humans. During replication, Okazaki fragment synthesis relies on frequent RNA primer placement, providing one of the most prominent forms of covalent RNA-DNA strands in vivo The mechanism of Okazaki fragment maturation, which involves RNA removal and subsequent DNA replacement, in bacteria lacking RNase HI remains unclear. In this work, we reconstituted repair of a linear model Okazaki fragment in vitro using purified recombinant enzymes from Bacillus subtilis We showed that RNase HII and HIII are capable of incision on Okazaki fragments in vitro and that both enzymes show mild stimulation by single-stranded DNA binding protein (SSB). We also showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation in vitro Furthermore, we found that YpcP is a 5' to 3' nuclease that can act on a wide variety of RNA- and DNA-containing substrates and exhibits preference for degrading RNA in model Okazaki fragments. Together, our data showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation, whereas YpcP also contributes to the removal of RNA from an Okazaki fragment in vitro IMPORTANCE All cells are required to resolve the different types of RNA-DNA hybrids that form in vivo When RNA-DNA hybrids persist, cells experience an increase in mutation rate and problems with DNA replication. Okazaki fragment synthesis on the lagging strand requires an RNA primer to begin synthesis of each fragment. The mechanism of RNA removal from Okazaki fragments remains unknown in bacteria that lack RNase HI. We examined Okazaki fragment processing in vitro and found that RNase HIII in conjunction with DNA polymerase I represent the most efficient repair pathway. We also assessed the contribution of YpcP and found that YpcP is a 5' to 3' exonuclease that prefers RNA substrates with activity on Okazaki and flap substrates in vitro.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Ribonucleases/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , DNA Polimerase I/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleases/genética , Ribonucleases/isolamento & purificação
16.
Biochemistry ; 58(6): 575-581, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30557012

RESUMO

The PX motif of DNA is a four-stranded structure in which two parallel juxtaposed double-helical domains are fused by crossovers at every point where the strands approach each other. Consequently, its twist and writhe are approximately half of those of conventional DNA. This property has been shown to relax supercoiled plasmid DNA under circumstances in which head-to-head homology exists within the plasmid; the homology can be either complete homology or every-other-half-turn homology, known as PX homology. It is clearly of interest to establish whether the cell contains proteins that interact with this unusual and possibly functional motif. We have examined Escherichia coli extracts to seek such a protein. We find by gel mobility studies that the PX motif is apparently bound by a cellular component. Fractionation of this binding activity reveals that the component is DNA polymerase I (Pol I). Although the PX motif binds to Pol I, we find that PX-DNA is not able to serve as a substrate for the extension of a shortened strand. We cannot say at this time whether the binding is a coincidence or whether it represents an activity of Pol I that is currently unknown. We have modeled the interaction of Pol I and PX-DNA using symmetry considerations and molecular dynamics.


Assuntos
DNA Polimerase I/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Motivos de Nucleotídeos , DNA Polimerase I/química , Replicação do DNA , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Modelos Moleculares , Conformação Proteica
17.
Org Biomol Chem ; 17(2): 290-301, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30543241

RESUMO

We developed a versatile access to a series of 4-substituted imidazole 2'-deoxynucleoside triphosphate bearing functionalized phenyl or pyrimidinyl rings. 4-Iodo-1H-imidazole was enzymatically converted into the corresponding 2'-deoxynucleoside, which was then chemically derived into its 5'-triphosphate, followed by 4-arylation via Suzuki-Miyaura coupling using (hetero)arylboronic acids. Both KF (exo-) and Deep Vent (exo-) DNA polymerases incorporated these modified nucleotides in primer-extension assays, adenine being the preferred pairing partner in the template. The 4-(3-aminophenyl)imidazole derivative (3APh) was the most efficiently inserted opposite A by KF (exo-) with only a 37-fold lower efficiency (Vmax/KM) than that of the correct dTTP. No further extension occurred after the incorporation of a single aryl-imidazole nucleotide. Interestingly, the aryl-imidazole dNTPs were found to undergo successive incorporation by calf thymus terminal deoxynucleotidyl transferase with different tailing efficiencies among this series and with a marked preference for 2APyr polymerization.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleosídeos/metabolismo , Imidazóis/metabolismo , Polifosfatos/metabolismo , Pirimidinas/metabolismo , Animais , Sequência de Bases , Bovinos , DNA Polimerase I/metabolismo , Desoxirribonucleosídeos/síntese química , Desoxirribonucleosídeos/química , Imidazóis/síntese química , Imidazóis/química , Polimerização , Polifosfatos/síntese química , Polifosfatos/química , Pirimidinas/síntese química , Pirimidinas/química
18.
J Bacteriol ; 201(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30530516

RESUMO

Escherichia coli has an ability to assemble DNA fragments with homologous overlapping sequences of 15 to 40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA cloning technology. However, the molecular mechanism by which E. coli accomplishes such cloning is still unknown. In this study, we provide evidence that the in vivo cloning of E. coli is independent of both RecA and RecET recombinases but is dependent on XthA, a 3' to 5' exonuclease. Here, in vivo cloning of E. coli by XthA is referred to as in vivo E. coli cloning (iVEC). We also show that iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3' ends of linear DNA fragments that are introduced into E. coli cells, resulting in exposure of the single-stranded 5' overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development of in vivo DNA cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC.IMPORTANCE Cloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, an in vitro recombination system for DNA cloning was shown to enable the joining of multiple DNA fragments at once. Interestingly, E. coli potentially assembles multiple linear DNA fragments that are introduced into the cell. Improved protocols for this in vivo cloning have realized a high level of usability, comparable to that by in vitro recombination reactions. However, the mechanism of in vivo cloning is highly controversial. Here, we clarified the fundamental mechanism underlying in vivo cloning and also constructed a strain that was optimized for in vivo cloning. Additionally, we streamlined the procedure of in vivo cloning by using a single microcentrifuge tube.


Assuntos
DNA Bacteriano/metabolismo , Escherichia coli/enzimologia , Exodesoxirribonucleases/metabolismo , Recombinação Genética , Clonagem Molecular , DNA Polimerase I/metabolismo , DNA Bacteriano/genética , Escherichia coli/metabolismo , Hibridização de Ácido Nucleico , Transformação Genética
19.
Curr Opin Struct Biol ; 53: 159-168, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30292863

RESUMO

Bacterial replisomes are dynamic multiprotein DNA replication machines that are inherently difficult for structural studies. However, breakthroughs continue to come. The structures of Escherichia coli DNA polymerase III (core)-clamp-DNA subcomplexes solved by single-particle cryo-electron microscopy in both polymerization and proofreading modes and the discovery of the stochastic nature of the bacterial replisomes represent notable progress. The structures reveal an intricate interaction network in the polymerase-clamp subassembly, providing insights on how replisomes may work. Meantime, ensemble and single-molecule functional assays and fluorescence microscopy show that the bacterial replisomes can work in a decoupled and uncoordinated way, with polymerases quickly exchanging and both leading-strand and lagging-strand polymerases and the helicase working independently, contradictory to the elegant textbook view of a highly coordinated machine.


Assuntos
Replicação do DNA , Escherichia coli , Complexos Multienzimáticos , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Bacteriófago T7/enzimologia , Bacteriófago T7/genética , DNA Helicases/química , DNA Helicases/metabolismo , DNA Ligases/química , DNA Ligases/metabolismo , DNA Polimerase I/química , DNA Polimerase I/metabolismo , DNA Polimerase III/química , DNA Polimerase III/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Helicobacter pylori/enzimologia , Helicobacter pylori/genética , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo
20.
DNA Repair (Amst) ; 70: 10-17, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30098577

RESUMO

Functioning DNA repair capabilities are vital for organisms to ensure that the biological information is preserved and correctly propagated. Disruptions in DNA repair pathways can result in the accumulation of DNA mutations, which may lead to onset of complex disease such as cancer. The discovery and characterization of cancer-related biomarkers may allow early diagnosis and targeted treatment, which could significantly contribute to the survival rates of cancer patients. To this end, we have applied a hypothesis driven bioinformatics approach to identify biomarkers related to 25 different DNA repair enzymes, in combination with structural analysis of six selected missense mutations of newly discovered SNPs that are associated with cancer phenotypes. Our search on 8 distinct cancer databases uncovered 43 missense SNPs that statistically significantly associated at least one phenotype. Moreover, nine of these missense SNPs are statistically significantly associated with two or more cancers. In addition, we have performed classical molecular dynamics to characterize the impact of rs10018786 on POLN, which results in the M310 L Pol ν variant, and rs3218784 on POLI, which results in the I236 M Pol ι. Our results suggest that both of these cancer-associated variants result in noticeable structural and dynamical changes compared with their respective wild-type proteins.


Assuntos
Biomarcadores Tumorais/genética , Reparo do DNA/genética , Bases de Dados Genéticas , Fenótipo , DNA Polimerase I/química , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Simulação de Dinâmica Molecular , Mutação , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Conformação Proteica
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