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1.
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
2.
Gene ; 668: 246-251, 2018 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-29621587

RESUMO

The mechanism by which bta-miR-378 regulates bovine skeletal muscle-derived satellite cell (bMDSC) myogenesis remains unknown. In this study, stem-loop RT-PCR was used to assess bta-miR-378 expression during the proliferation and differentiation of bMDSCs. The results showed that bta-miR-378 expression did not obviously change during bMDSC proliferation but increased significantly when bMDSCs began to differentiate. Then, a bta-miR-378 mimic (bta-miR-378-M) and bta-miR-378 inhibitor (bta-miR-378-I) were transfected into bMDSCs to explore the effect of bta-miR-378 on bMDSC differentiation. Cell differentiation was detected using myosin heavy chain 3 immunofluorescence, myotube formation, and desmin and myogenin western blotting analyses. As expected, bta-miR-378-M enhanced bMDSC differentiation, whereas bta-miR-378-I had the opposite effect. Moreover, luciferase reporter and western blotting assays showed that bta-miR-378 directly targeted the 3'-untranslated regions of DNA polymerase alpha subunit B (POLA2) to regulate its protein expression. In summary, these data indicate that bta-miR-378 targets POLA2 to promote the differentiation of bMDSCs, which provides further insight into the biological functions of bta-miR-378 in bovines.


Assuntos
Bovinos/genética , MicroRNAs/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Animais , Bovinos/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Regulação da Expressão Gênica , Células Satélites de Músculo Esquelético/citologia
3.
DNA Repair (Amst) ; 64: 59-67, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29522920

RESUMO

Deamination of adenine can occur spontaneously under physiological conditions to generate the highly mutagenic lesion, deoxyinosine (hypoxanthine deoxyribonucleotide, dI). In DNA, dI preferably pairs with cytosine rather than thymine and results in A:T to G:C transition mutations after DNA replication. The deamination of adenine is enhanced by ROS from exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. In Escherichia coli, dI repair is initiated by endonuclease V (endo V; nfi gene product) nicking but a complete repair mechanism has yet to be elucidated. Using in vitro minimum component reconstitution assays, we previously showed that endo V, DNA polymerase I (pol I), and E. coli DNA ligase were sufficient to repair this dI lesions efficiently and that the 3'-5' exonuclease of pol I is essential. Here we employed a phagemid-based T-I substrate mimicking adenine deamination product to demonstrate pol I proofreading exonuclease is required by the endo V repair pathway both in vitro and in vivo. In vivo we found that the repair level of an nfi mutant (11%) was almost 8-fold lower than the wild type (87%). while the polA-D424A strain, a pol I mutant defective in 3'-5' exonuclease, showed a high repair level similar to wild type (both more than 80%). Using additional C-C mismatch as strand discrimination marker we found that the high level of dI removal in polA-D424A was due to strand loss (more than 60%) associated with incomplete repair. Thus, pol I proofreading exonuclease is the major function responsible for dI lesion removal after endoV nicking both in vitro and in vivo. Finally, using MALDI-TOF to analyze single-nucleotide extension product we show that the pol I proofreading exonuclease excises only 2-nt 5' upstream of endo V incision site further honing the role of pol I in the endoV dI dependent repair pathway.


Assuntos
Dano ao DNA , DNA Polimerase I/metabolismo , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Inosina/análogos & derivados , DNA/metabolismo , DNA Ligases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Inosina/metabolismo
4.
Biochem Biophys Res Commun ; 499(2): 170-176, 2018 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-29550479

RESUMO

Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5' to 3' exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol.


Assuntos
DNA Polimerase I/metabolismo , Replicação do DNA , Engenharia de Proteínas , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , DNA Polimerase I/química , Evolução Molecular Direcionada , Mutação/genética
5.
Methods Mol Biol ; 1681: 165-178, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29134594

RESUMO

Genomics has greatly transformed our understanding of phage biology; however, traditional methods of DNA isolation for whole genome sequencing have required phages to be grown to high titers in large-scale preparations, potentially selecting for only those phages that can grow efficiently under laboratory conditions. This may also select for mutations or deletions that enable more efficient growth in culture. The ability to sequence a bacteriophage genome from a single isolated plaque reduces these risks while decreasing the time and complexity of bacteriophage genome sequencing. A method of amplification and library preparation is described, utilizing Sequence Independent Single Primer Amplification (SISPA), that can be used for whole genome shotgun sequencing of bacteriophages from a single isolated plaque.


Assuntos
Bacteriófagos/genética , Primers do DNA/metabolismo , Reação em Cadeia da Polimerase/métodos , Ensaio de Placa Viral , Sequenciamento Completo do Genoma/métodos , Contaminação por DNA , DNA Polimerase I/metabolismo , Desnaturação de Ácido Nucleico , Ácidos Nucleicos/isolamento & purificação , Vírus de RNA/genética
6.
DNA Repair (Amst) ; 61: 63-75, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29223016

RESUMO

Proofreading and DNA repair are important factors in maintaining the high fidelity of genetic information during DNA replication. Herein, we designed a non-labeled and non-radio-isotopic simple method to measure proofreading. An oligonucleotide primer is annealed to a template DNA forming a mismatched site and is proofread by Klenow fragment of Escherichia coli DNA polymerase I (pol I) in the presence of all four dideoxyribonucleotide triphosphates. The proofreading excision products and re-synthesis products of single nucleotide extension are subjected to MALDI-TOF mass spectrometry (MS). The proofreading at the mismatched site is identified by the mass change of the primer. We examined proofreading of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus can be proofread efficiently. Internal single mismatches can also be proofread at different efficiencies, with the best correction for mismatches located 2-4-nucleotides from the primer terminus. For mismatches located 5-nucleotides from the primer terminus there was partial correction and extension. No significant proofreading was observed for mismatches located 6-9-nucleotides from the primer terminus. We also subjected primers containing 3' penultimate deoxyinosine (dI) lesions, which mimic endonuclease V nicked repair intermediates, to pol I repair assay. The results showed that T-I was a better substrate than G-I and A-I, however C-I was refractory to repair. The high resolution of MS results clearly demonstrated that all the penultimate T-I, G-I and A-I substrates had been excised last 2 dI-containing nucleotides by pol I before adding a correct ddNMP, however, pol I proofreading exonuclease tolerated the penultimate C-I mismatch allowing the primer to be extended by polymerase activity.


Assuntos
Reparo do DNA , Replicação do DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , DNA Polimerase I/metabolismo , Moldes Genéticos
7.
Open Biol ; 7(11)2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29167311

RESUMO

A dynamic multi-protein assembly known as the replisome is responsible for DNA synthesis in eukaryotic cells. In yeast, the hub protein Ctf4 bridges DNA helicase and DNA polymerase and recruits factors with roles in metabolic processes coupled to DNA replication. An important question in DNA replication is the extent to which the molecular architecture of the replisome is conserved between yeast and higher eukaryotes. Here, we describe the biochemical basis for the interaction of the human CTF4-orthologue AND-1 with DNA polymerase α (Pol α)/primase, the replicative polymerase that initiates DNA synthesis. AND-1 has maintained the trimeric structure of yeast Ctf4, driven by its conserved SepB domain. However, the primary interaction of AND-1 with Pol α/primase is mediated by its C-terminal HMG box, unique to mammalian AND-1, which binds the B subunit, at the same site targeted by the SV40 T-antigen for viral replication. In addition, we report a novel DNA-binding activity in AND-1, which might promote the correct positioning of Pol α/primase on the lagging-strand template at the replication fork. Our findings provide a biochemical basis for the specific interaction between two critical components of the human replisome, and indicate that important principles of replisome architecture have changed significantly in evolution.


Assuntos
DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Domínios HMG-Box , Sítios de Ligação , Biologia Computacional , Humanos , Modelos Moleculares , Ligação Proteica
8.
FEBS J ; 284(23): 4051-4065, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28986969

RESUMO

Non-canonical four-stranded G-quadruplex (G4) DNA structures can form in G-rich sequences that are widely distributed throughout the genome. The presence of G4 structures can impair DNA replication by hindering the progress of replicative polymerases (Pols), and failure to resolve these structures can lead to genetic instability. In the present study, we combined different approaches to address the question of whether and how Escherichia coli Pol I resolves G4 obstacles during DNA replication and/or repair. We found that E. coli Pol I-catalyzed DNA synthesis could be arrested by G4 structures at low protein concentrations and the degree of inhibition was strongly dependent on the stability of the G4 structures. Interestingly, at high protein concentrations, E. coli Pol I was able to overcome some kinds of G4 obstacles without the involvement of other molecules and could achieve complete replication of G4 DNA. Mechanistic studies suggested that multiple Pol I proteins might be implicated in G4 unfolding, and the disruption of G4 structures requires energy derived from dNTP hydrolysis. The present work not only reveals an unrealized function of E. coli Pol I, but also presents a possible mechanism by which G4 structures can be resolved during DNA replication and/or repair in E. coli.


Assuntos
DNA Polimerase I/metabolismo , Replicação do DNA , Proteínas de Escherichia coli/metabolismo , Quadruplex G , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Modelos Genéticos , Modelos Moleculares , Conformação de Ácido Nucleico
9.
Nucleic Acids Res ; 45(21): e175, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29036327

RESUMO

We describe a method for measuring the single-turnover incorporation kinetics of non-fluorescent native nucleotides by DNA polymerases. Time-lapse total internal reflection fluorescence (TIRF) microscopy is used to directly measure the kinetics of single-base nucleotide incorporation into primed DNA templates covalently attached to the surface of a glass coverslip using a fixed ratio of a native nucleotide and a corresponding fluorescently labeled nucleotide over a series of total nucleotide concentrations. The presence of a labeled nucleotide allows for the kinetics of competitive incorporation reactions by DNA polymerase to be monitored. The single-turnover catalytic rate constants and Michaelis constants of the incorporation of the native nucleotides can be determined by modeling the kinetics of the parallel competitive reactions. Our method enables the measurements of the kinetics parameters of incorporation of native or other non-fluorescent nucleotides without using a rapid stopped-flow or quench-flow instrument and the generally more involved and less quantitative post-reaction analysis of the reaction products. As a demonstration of our method, we systematically determined the single-turnover incorporation kinetics of all four native nucleotides and a set of Cy3-labeled nucleotides by the Klenow fragment of Escherichia coli DNA polymerase I.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Microscopia de Fluorescência , Nucleotídeos/metabolismo , Imagem com Lapso de Tempo/métodos , DNA Polimerase I/metabolismo , Cinética
10.
Chem Res Toxicol ; 30(11): 2033-2043, 2017 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-29053918

RESUMO

Replicative DNA polymerases are able to discriminate between very similar substrates with high accuracy. One mechanism by which E. coli DNA polymerase I checks for Watson-Crick geometry is through a hydrogen bonding fork between Arg668 and the incoming dNTP and the minor groove of the primer terminus. The importance of the Arg-fork was examined by disrupting it with either a guanine to 3-deazaguanine substitution at the primer terminus or the use of a carbocyclic deoxyribose analog of dUTP. Using thio-substituted dNTPs and differential quench techniques, we determined that when the Arg-fork was disrupted, the rate-limiting step changed from a conformational change to phosphodiester bond formation. This result indicates that Arg668 is involved in the phosphoryl transfer step. We examined the role of the Arg-fork in the replication of four DNA damaged templates, O6-methylguanine (O6-mG), 8-oxo-7,8-dihydroguanine (oxoG), O2-[4-(3-pyridyl)-4-oxobutyl]thymine (O2-POB-T), and N2-[(7S,8R,9S,10R)-7,8,9,10-tetrahydro-8,9,10-trihydroxybenzo[a]pyren-7-yl]-guanine (N2-BP-G). In general, the guanine to 3-deazaguanine substitution caused a decrease in kpol that was proportional to kpol over five orders of magnitude. The linear relationship indicates that the Arg668-fork helps catalyze phosphoryl transfer by the same mechanism with all the substrates. Exceptions to the linear relationship were the incorporations of dTTP opposite G, oxoG, and O6mG, which showed large decreases in kpol, similar to that exhibited by the Watson-Crick base pairs. It was proposed that the incorporation of dTTP opposite G, oxoG, and O6mG occurred via Watson-Crick-like structures.


Assuntos
Dano ao DNA , DNA Polimerase I/metabolismo , Replicação do DNA , DNA Bacteriano/genética , Escherichia coli/enzimologia , Pareamento de Bases , Domínio Catalítico , DNA Polimerase I/química , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fosforilação
11.
Proc Natl Acad Sci U S A ; 114(44): 11733-11738, 2017 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-29078353

RESUMO

Replicative DNA polymerases misincorporate ribonucleoside triphosphates (rNTPs) into DNA approximately once every 2,000 base pairs synthesized. Ribonucleotide excision repair (RER) removes ribonucleoside monophosphates (rNMPs) from genomic DNA, replacing the error with the appropriate deoxyribonucleoside triphosphate (dNTP). Ribonucleotides represent a major threat to genome integrity with the potential to cause strand breaks. Furthermore, it has been shown in the bacterium Bacillus subtilis that loss of RER increases spontaneous mutagenesis. Despite the high rNTP error rate and the effect on genome integrity, the mechanism underlying mutagenesis in RER-deficient bacterial cells remains unknown. We performed mutation accumulation lines and genome-wide mutational profiling of B. subtilis lacking RNase HII, the enzyme that incises at single rNMP residues initiating RER. We show that loss of RER in B. subtilis causes strand- and sequence-context-dependent GC → AT transitions. Using purified proteins, we show that the replicative polymerase DnaE is mutagenic within the sequence context identified in RER-deficient cells. We also found that DnaE does not perform strand displacement synthesis. Given the use of nucleotide excision repair (NER) as a backup pathway for RER in RNase HII-deficient cells and the known mutagenic profile of DnaE, we propose that misincorporated ribonucleotides are removed by NER followed by error-prone resynthesis with DnaE.


Assuntos
Bacillus subtilis/genética , DNA Bacteriano/genética , Ribonucleotídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/fisiologia , Modelos Biológicos , Mutagênese , Mutação , Ribonuclease H/genética , Ribonuclease H/metabolismo
12.
Protein J ; 36(6): 453-460, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28932939

RESUMO

A structural and kinetic characterization of a fragment of the HoLaMa DNA polymerase is presented here. In particular, a truncated form of HoLaMa, devoid of a consistent portion of the thumb domain, was isolated and purified. This HoLaMa fragment, denoted as ΔNter-HoLaMa, is surprisingly competent in catalyzing DNA extension, albeit featuring a kcat one order of magnitude lower than the corresponding kinetic constant of its full-length counterpart. The conformational rearrangements, if any, of enzyme tryptophanes triggered by DNA binding or extension were assayed under pre-steady-state conditions. The fluorescence of HoLaMa tryptophanes was found to significantly change upon DNA binding and extension. On the contrary, no fluorescence changes of ΔNter-HoLaMa tryptophanes were detected under the same conditions, suggesting that major conformational transitions are not required for DNA binding or extension by this truncated DNA polymerase.


Assuntos
Domínio Catalítico , DNA Polimerase I , Domínio Catalítico/genética , Domínio Catalítico/fisiologia , DNA Polimerase I/química , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Exonucleases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
Nucleic Acids Res ; 45(16): 9455-9466, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28934486

RESUMO

The CST (CTC1-STN1-TEN1) complex mediates critical functions in maintaining telomere DNA and overcoming genome-wide replication stress. A conserved biochemical function of the CST complex is its primase-Pol α (PP) stimulatory activity. In this report, we demonstrate the ability of purified human STN1 alone to promote PP activity in vitro. We show that this regulation is mediated primarily by the N-terminal OB fold of STN1, but does not require the DNA-binding activity of this domain. Rather, we observed a strong correlation between the PP-stimulatory activity of STN1 variants and their abilities to bind POLA2. Remarkably, the main binding target of STN1 in POLA2 is the latter's central OB fold domain. In the substrate-free structure of PP, this domain is positioned so as to block nucleic acid entry to the Pol α active site. Thus the STN1-POLA2 interaction may promote the necessary conformational change for nucleic acid delivery to Pol α and subsequent DNA synthesis. A disease-causing mutation in human STN1 engenders a selective defect in POLA2-binding and PP stimulation, indicating that these activities are critical for the in vivo function of STN1. Our findings have implications for the molecular mechanisms of PP, STN1 and STN1-related molecular pathology.


Assuntos
DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Sítios de Ligação , DNA/metabolismo , DNA Polimerase I/química , DNA Polimerase I/genética , DNA Primase/química , DNA Primase/genética , Humanos , Mutação Puntual , Domínios Proteicos , Subunidades Proteicas , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/genética
14.
Nucleic Acids Res ; 45(16): 9595-9610, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28934494

RESUMO

At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNA-binding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants-urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.


Assuntos
DNA Polimerase I/metabolismo , Geobacillus/enzimologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Domínio Catalítico , Clonagem Molecular , DNA/metabolismo , DNA Polimerase I/antagonistas & inibidores , DNA Polimerase I/genética , Inibidores Enzimáticos/farmacologia , Genoma Humano , Geobacillus/genética , Geobacillus stearothermophilus/enzimologia , Geobacillus stearothermophilus/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Estabilidade Proteica , Pyrococcus abyssi/genética , Proteínas Recombinantes de Fusão/metabolismo , Sulfolobus/genética
15.
Mol Cell ; 67(5): 867-881.e7, 2017 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-28757209

RESUMO

Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.


Assuntos
Proteína BRCA2/metabolismo , Replicação do DNA , DNA/biossíntese , Rad51 Recombinase/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Proteína BRCA2/genética , Sítios de Ligação , DNA/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase I/metabolismo , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Feminino , Instabilidade Genômica , Humanos , Proteína Homóloga a MRE11 , Masculino , Mutação , Ligação Proteica , Rad51 Recombinase/genética , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Proteínas de Xenopus/genética , Xenopus laevis/genética
16.
Proc Natl Acad Sci U S A ; 114(36): 9605-9610, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28827350

RESUMO

Noncanonical DNA structures that stall DNA replication can cause errors in genomic DNA. Here, we investigated how the noncanonical structures formed by sequences in genes associated with a number of diseases impacted DNA polymerization by the Klenow fragment of DNA polymerase. Replication of a DNA sequence forming an i-motif from a telomere, hypoxia-induced transcription factor, and an insulin-linked polymorphic region was effectively inhibited. On the other hand, replication of a mixed-type G-quadruplex (G4) from a telomere was less inhibited than that of the antiparallel type or parallel type. Interestingly, the i-motif was a better inhibitor of replication than were mixed-type G4s or hairpin structures, even though all had similar thermodynamic stabilities. These results indicate that both the stability and topology of structures formed in DNA templates impact the processivity of a DNA polymerase. This suggests that i-motif formation may trigger genomic instability by stalling the replication of DNA, causing intractable diseases.


Assuntos
DNA Polimerase I/metabolismo , DNA/química , DNA/metabolismo , Conformação de Ácido Nucleico , Replicação do DNA , Quadruplex G , Instabilidade Genômica , Humanos , Modelos Biológicos , Modelos Moleculares , Motivos de Nucleotídeos , Telômero/metabolismo , Termodinâmica
17.
J Immunol ; 199(7): 2316-2322, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28807995

RESUMO

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) display unique aggressive behavior, invading the articular cartilage and promoting inflammation. Using an integrative analysis of RA risk alleles, the transcriptome and methylome in RA FLS, we recently identified the limb bud and heart development (LBH) gene as a key dysregulated gene in RA and other autoimmune diseases. Although some evidence suggests that LBH could modulate the cell cycle, the precise mechanism is unknown and its impact on inflammation in vivo has not been defined. Our cell cycle analysis studies show that LBH deficiency in FLS leads to S-phase arrest and failure to progress through the cell cycle. LBH-deficient FLS had increased DNA damage and reduced expression of the catalytic subunit of DNA polymerase α. Decreased DNA polymerase α was followed by checkpoint arrest due to phosphorylation of checkpoint kinase 1. Because DNA fragments can increase arthritis severity in preclinical models, we then explored the effect of LBH deficiency in the K/BxN serum transfer model. Lbh knockout exacerbated disease severity, which is associated with elevated levels of IL-1ß and checkpoint kinase 1 phosphorylation. These studies indicate that LBH deficiency induces S-phase arrest that, in turn, exacerbates inflammation. Because LBH gene variants are associated with type I diabetes mellitus, systemic lupus erythematosus, RA, and celiac disease, these results suggest a general mechanism that could contribute to immune-mediated diseases.


Assuntos
Artrite Reumatoide/genética , Ciclo Celular/genética , Proteínas Nucleares/genética , Sinoviócitos/imunologia , Animais , Artrite Experimental , Artrite Reumatoide/imunologia , Artrite Reumatoide/fisiopatologia , Células Cultivadas , Quinase 1 do Ponto de Checagem/genética , Dano ao DNA , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Regulação da Expressão Gênica , Genes cdc , Humanos , Interleucina-1beta/biossíntese , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Fosforilação , Transdução de Sinais
18.
Nucleic Acids Res ; 45(12): 7261-7275, 2017 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-28510759

RESUMO

Mcm10 is an essential eukaryotic factor required for DNA replication. The replication fork helicase is composed of Cdc45, Mcm2-7 and GINS (CMG). DDK is an S-phase-specific kinase required for replication initiation, and the DNA primase-polymerase in eukaryotes is pol α. Mcm10 forms oligomers in vitro, mediated by the coiled-coil domain at the N-terminal region of the protein. We characterized an Mcm10 mutant at the N-terminal Domain (NTD), Mcm10-4A, defective for self-interaction. We found that the Mcm10-4A mutant was defective for stimulating DDK phosphorylation of Mcm2, binding to eighty-nucleotide ssDNA, and recruiting pol α to Mcm2-7 in vitro. Expression of wild-type levels of mcm10-4A resulted in severe growth and DNA replication defects in budding yeast cells, with diminished DDK phosphorylation of Mcm2. We then expressed the mcm10-4A in mcm5-bob1 mutant cells to bypass the defects mediated by diminished stimulation of DDK phosphorylation of Mcm2. Expression of wild-type levels of mcm10-4A in mcm5-bob1 mutant cells resulted in severe growth and DNA replication defects, along with diminished RPA signal at replication origins. We also detected diminished GINS and pol-α recruitment to the Mcm2-7 complex. We conclude that an intact Mcm10 coiled-coil interaction surface is important for origin melting, helicase assembly, and the recruitment of pol α to Mcm2-7.


Assuntos
DNA Polimerase I/genética , Replicação do DNA , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Polimerase I/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Cinética , Camundongos , Proteínas de Manutenção de Minicromossomo/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo
19.
Chem Res Toxicol ; 30(5): 1168-1176, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28402640

RESUMO

Benzo[a]pyrene, a potent human carcinogen, is metabolized in vivo to a diol epoxide that reacts with the N2-position of guanine to produce N2-BP-dG adducts. These adducts are mutagenic causing G to T transversions. These adducts block replicative polymerases but can be bypassed by the Y-family translesion synthesis polymerases. The mechanisms by which mutagenic bypass occurs is not well-known. We have evaluated base pairing structures using atomic substitution of the dNTP with two stereoisomers, 2'-deoxy-N-[(7R,8S,9R,10S)-7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine and 2'-deoxy-N-[(7S,8R,9S,10R)-7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine. We have examined the kinetics of incorporation of 1-deaza-dATP, 7-deaza-dATP, 2'-deoxyinosine triphosphate, and 7-deaza-dGTP, analogues of dATP and dGTP in which single atoms are changed. Changes in rate will occur if that atom provided a critical interaction in the transition state of the reaction. We examined two polymerases, Escherichia coli DNA polymerase I (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4), as models of a high fidelity and TLS polymerase, respectively. We found that with Kf, substitution of the nitrogens on the Watson-Crick face of the dNTPs resulted in decreased rate of reactions. This result is consistent with a Hoogsteen base pair in which the template N2-BP-dG flipped from the anti to syn conformation. With Dpo4, while the substitution did not affect the rate of reaction, the amplitude of the reaction decreased with all substitutions. This result suggests that Dpo4 bypasses N2-BP-dG via Hoogsteen base pairs but that the flipped nucleotide can be either the dNTP or the template.


Assuntos
Benzopirenos/metabolismo , Adutos de DNA , DNA Polimerase I/metabolismo , DNA Polimerase beta/metabolismo , Replicação do DNA , Desoxiguanosina/análogos & derivados , Escherichia coli/enzimologia , Sulfolobus solfataricus/enzimologia , Pareamento de Bases , Catálise , Desoxiguanosina/metabolismo
20.
J Biol Chem ; 292(23): 9627-9636, 2017 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-28381552

RESUMO

DNA replication in eukaryotic cells is performed by a multiprotein complex called the replisome, which consists of helicases, polymerases, and adaptor molecules. Human acidic nucleoplasmic DNA-binding protein 1 (AND-1), also known as WD repeat and high mobility group (HMG)-box DNA-binding protein 1 (WDHD1), is an adaptor molecule crucial for DNA replication. Although structural information for the AND-1 yeast ortholog is available, the mechanistic details for how human AND-1 protein anchors the lagging-strand DNA polymerase α (pol α) to the DNA helicase complex (Cdc45-MCM2-7-GINS, CMG) await elucidation. Here, we report the structures of the N-terminal WD40 and SepB domains of human AND-1, as well as a biochemical analysis of the C-terminal HMG domain. We show that AND-1 exists as a homotrimer mediated by the SepB domain. Mutant study results suggested that a positively charged groove within the SepB domain provides binding sites for pol α. Different from its ortholog protein in budding yeast, human AND-1 is recruited to the CMG complex, mediated by unknown participants other than Go Ichi Ni San. In addition, we show that AND-1 binds to DNA in vitro, using its C-terminal HMG domain. In conclusion, our findings provide important insights into the mechanistic details of human AND-1 function, advancing our understanding of replisome formation during eukaryotic replication.


Assuntos
DNA Helicases/química , DNA Polimerase I/química , Proteínas de Ligação a DNA/química , DNA/química , Complexos Multienzimáticos/química , Multimerização Proteica , DNA/biossíntese , DNA/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Domínios Proteicos
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