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1.
Nucleic Acids Res ; 49(4): 2179-2191, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33533925

RESUMO

Replication forks often stall at damaged DNA. To overcome these obstructions and complete the DNA duplication in a timely fashion, replication can be restarted downstream of the DNA lesion. In mammalian cells, this repriming of replication can be achieved through the activities of primase and polymerase PrimPol. PrimPol is stimulated in DNA synthesis through interaction with PolDIP2, however the exact mechanism of this PolDIP2-dependent stimulation is still unclear. Here, we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol's enhanced processivity. PolDIP2 increases primer-template and dNTP binding affinities of PrimPol, which concomitantly enhances its nucleotide incorporation efficiency. This stimulation is dependent on a unique arginine cluster in PolDIP2. Since the polymerase activity of PrimPol alone is very limited, this mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, might be critical for the in vivo function of PrimPol in tolerating DNA lesions at physiological nucleotide concentrations.


Assuntos
Arginina/química , DNA Primase/química , DNA Polimerase Dirigida por DNA/química , DNA/biossíntese , Enzimas Multifuncionais/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Motivos de Aminoácidos , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Modelos Moleculares , Enzimas Multifuncionais/metabolismo , Ligação Proteica
2.
Nucleic Acids Res ; 49(4): 2065-2084, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33555350

RESUMO

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.


Assuntos
Replicação do DNA , DNA/química , DNA/metabolismo , Quadruplex G , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Linhagem Celular , DNA Polimerase Dirigida por DNA/metabolismo , Genes myc , Humanos , Modelos Moleculares , Mutação , Motivos de Nucleotídeos , Nucleotidiltransferases/genética , Ligação Proteica
3.
Nat Commun ; 12(1): 1044, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594055

RESUMO

CrAssphage is the most abundant human-associated virus and the founding member of a large group of bacteriophages, discovered in animal-associated and environmental metagenomes, that infect bacteria of the phylum Bacteroidetes. We analyze 4907 Circular Metagenome Assembled Genomes (cMAGs) of putative viruses from human gut microbiomes and identify nearly 600 genomes of crAss-like phages that account for nearly 87% of the DNA reads mapped to these cMAGs. Phylogenetic analysis of conserved genes demonstrates the monophyly of crAss-like phages, a putative virus order, and of 5 branches, potential families within that order, two of which have not been identified previously. The phage genomes in one of these families are almost twofold larger than the crAssphage genome (145-192 kilobases), with high density of self-splicing introns and inteins. Many crAss-like phages encode suppressor tRNAs that enable read-through of UGA or UAG stop-codons, mostly, in late phage genes. A distinct feature of the crAss-like phages is the recurrent switch of the phage DNA polymerase type between A and B families. Thus, comparative genomic analysis of the expanded assemblage of crAss-like phages reveals aspects of genome architecture and expression as well as phage biology that were not apparent from the previous work on phage genomics.


Assuntos
Bacteriófagos/genética , Microbioma Gastrointestinal/genética , Genoma Viral , Metagenoma , Códon/genética , Sequência Conservada , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Inteínas , Íntrons/genética , Fases de Leitura Aberta/genética , Filogenia , Processamento de RNA/genética , Transcrição Genética , /genética
4.
Nat Commun ; 12(1): 923, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568651

RESUMO

Replication forks restarted by homologous recombination are error prone and replicate both strands semi-conservatively using Pol δ. Here, we use polymerase usage sequencing to visualize in vivo replication dynamics of HR-restarted forks at an S. pombe replication barrier, RTS1, and model replication by Monte Carlo simulation. We show that HR-restarted forks synthesise both strands with Pol δ for up to 30 kb without maturing to a δ/ε configuration and that Pol α is not used significantly on either strand, suggesting the lagging strand template remains as a gap that is filled in by Pol δ later. We further demonstrate that HR-restarted forks progress uninterrupted through a fork barrier that arrests canonical forks. Finally, by manipulating lagging strand resection during HR-restart by deleting pku70, we show that the leading strand initiates replication at the same position, signifying the stability of the 3' single strand in the context of increased resection.


Assuntos
Replicação do DNA , Recombinação Homóloga , Schizosaccharomyces/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
5.
Nat Commun ; 12(1): 482, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33473124

RESUMO

DNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1Rad27 Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1Cdc9 high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM.


Assuntos
Replicação do DNA/fisiologia , DNA Fúngico/metabolismo , DNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferases/metabolismo , DNA Ligase Dependente de ATP/metabolismo , DNA Ligases , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Endonucleases Flap/metabolismo , Proteínas de Membrana/metabolismo , Mutagênese , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Biochemistry ; 60(1): 1-5, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33356161

RESUMO

A recently described DNA polymerase ribozyme, obtained by in vitro evolution, provides the opportunity to investigate mechanistic features of RNA catalysis using methods that previously had only been applied to DNA polymerase proteins. Insight can be gained into the transition state of the DNA polymerization reaction by studying the behavior of various ß,γ-bridging substituted methylene (CXY; X, Y = H, halo, methyl) or imido (NH) dNTP analogues that differ with regard to the pKa4 of the bisphosphonate or imidodiphosphate leaving group. The apparent rate constant (kpol) of the polymerase ribozyme was determined for analogues of dGTP and dCTP that span a broad range of acidities for the leaving group, ranging from 7.8 for the CF2-bisphosphonate to 11.6 for the CHCH3-bisphosphonate. A Brønsted plot of log(kpol) versus pKa4 of the leaving group demonstrates linear free energy relationships (LFERs) for dihalo-, monohalo-, and non-halogen-substituted analogues of the dNTPs, with negative slopes, as has been observed for DNA polymerase proteins. The unsubstituted dNTPs have a faster catalytic rate than would be predicted from consideration of the linear free energy relationship alone, presumably due to a relatively more favorable interaction of the ß,γ-bridging oxygen within the active site. Although the DNA polymerase ribozyme is considerably slower than DNA polymerase proteins, it exhibits a similar LFER fingerprint, suggesting mechanistic commonality pertaining to the buildup of negative charge in the transition state, despite the very different chemical compositions of the two catalysts.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/química , Desoxirribonucleotídeos/química , Polifosfatos/química , RNA Catalítico/metabolismo , Humanos , Cinética , Polimerização , RNA Catalítico/química
7.
Subcell Biochem ; 96: 233-258, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33252731

RESUMO

In all cell types, a multi-protein machinery is required to accurately duplicate the large duplex DNA genome. This central life process requires five core replisome factors in all cellular life forms studied thus far. Unexpectedly, three of the five core replisome factors have no common ancestor between bacteria and eukaryotes. Accordingly, the replisome machines of bacteria and eukaryotes have important distinctions in the way that they are organized and function. This chapter outlines the major replication proteins that perform DNA duplication at replication forks, with particular attention to differences and similarities in the strategies used by eukaryotes and bacteria.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Bactérias/enzimologia , Bactérias/genética , Eucariotos/enzimologia , Eucariotos/genética
8.
Mol Cell ; 81(3): 442-458.e9, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33321094

RESUMO

Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.


Assuntos
Cromatina/metabolismo , Quebras de DNA de Cadeia Simples , Reparo do DNA , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Cromatina/genética , DNA Polimerase Dirigida por DNA/metabolismo , Feminino , Humanos , Antígeno Nuclear de Célula em Proliferação/genética , Especificidade por Substrato , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Xenopus laevis
9.
Int J Mol Sci ; 21(24)2020 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-33322195

RESUMO

The CMG complex (Cdc45, Mcm2-7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.


Assuntos
DNA Helicases/metabolismo , DNA Polimerase II/metabolismo , Replicação do DNA/genética , Proteínas Nucleares/metabolismo , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , DNA Polimerase II/genética , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Mudança da Fase de Leitura do Gene Ribossômico/genética , Mudança da Fase de Leitura do Gene Ribossômico/efeitos da radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Mutagênese , Mutação , Taxa de Mutação , Proteínas Nucleares/genética , Ligação Proteica , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinação Genética/efeitos da radiação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Mutações Sintéticas Letais/genética
10.
Proc Natl Acad Sci U S A ; 117(52): 33436-33445, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33376220

RESUMO

Fanconi anemia (FA) is caused by defects in cellular responses to DNA crosslinking damage and replication stress. Given the constant occurrence of endogenous DNA damage and replication fork stress, it is unclear why complete deletion of FA genes does not have a major impact on cell proliferation and germ-line FA patients are able to progress through development well into their adulthood. To identify potential cellular mechanisms that compensate for the FA deficiency, we performed dropout screens in FA mutant cells with a whole genome guide RNA library. This uncovered a comprehensive genome-wide profile of FA pathway synthetic lethality, including POLI and CDK4 As little is known of the cellular function of DNA polymerase iota (Pol ι), we focused on its role in the loss-of-function FA knockout mutants. Loss of both FA pathway function and Pol ι leads to synthetic defects in cell proliferation and cell survival, and an increase in DNA damage accumulation. Furthermore, FA-deficient cells depend on the function of Pol ι to resume replication upon replication fork stalling. Our results reveal a critical role for Pol ι in DNA repair and replication fork restart and suggest Pol ι as a target for therapeutic intervention in malignancies carrying an FA gene mutation.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Anemia de Fanconi/enzimologia , Estresse Fisiológico , Sistemas CRISPR-Cas/genética , Quinase 4 Dependente de Ciclina , Dano ao DNA , Genoma Humano , Células HCT116 , Humanos , Mutação/genética , Mutações Sintéticas Letais/genética
11.
Nat Commun ; 11(1): 5379, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097731

RESUMO

Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3'-5' exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of E. coli replicative DNA polymerase Pol III. We identify structures for all intermediates, in which the transitioning primer strand is stabilized by conserved Pol III residues along the fingers, thumb and exonuclease domains. We demonstrate switching kinetics on a tens of milliseconds timescale and unveil a complete pol-to-exo switching mechanism, validated by targeted mutational experiments.


Assuntos
Replicação do DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Polimerização , DNA/química , DNA Polimerase III/metabolismo , Primers do DNA , DNA Polimerase Dirigida por DNA/química , Escherichia coli/metabolismo , Exonucleases/metabolismo , Cinética , Modelos Moleculares , Conformação Proteica
12.
Proc Natl Acad Sci U S A ; 117(41): 25494-25504, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32999062

RESUMO

During DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites, as well as minor-groove and exocyclic guanine adducts. Lesion bypass is accomplished using a unique protein-template mechanism in which the templating base is evicted from the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain of Rev1. To understand the protein-template mechanism at an atomic level, we employed a combination of time-lapse X-ray crystallography, molecular dynamics simulations, and DNA enzymology on the Saccharomyces cerevisiae Rev1 protein. We find that Rev1 evicts the templating base from the DNA helix prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, although this is not coupled to large structural changes in Rev1 like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two monophosphates, which drives the reaction in the forward direction and prevents pyrophosphorolysis. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These postcatalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.


Assuntos
Reparo do DNA , Replicação do DNA/fisiologia , DNA/metabolismo , Nucleotidiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA/química , Dano ao DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Simulação de Dinâmica Molecular , Nucleotidiltransferases/genética , Proteínas de Saccharomyces cerevisiae/genética
13.
Mol Cell ; 80(1): 114-126.e8, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32916094

RESUMO

DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here, we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, redefining the architecture of the active eukaryotic replisome.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Células Eucarióticas/metabolismo , Complexos Multienzimáticos/metabolismo , Núcleo Celular/metabolismo , Cinética , Modelos Biológicos , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Fatores de Tempo
14.
Nucleic Acids Res ; 48(19): 10986-10997, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-32997110

RESUMO

During DNA replication, the presence of 8-oxoguanine (8-oxoG) lesions in the template strand cause the high-fidelity (HiFi) DNA polymerase (Pol) to stall. An early response to 8-oxoG lesions involves 'on-the-fly' translesion synthesis (TLS), in which a specialized TLS Pol is recruited and replaces the stalled HiFi Pol for lesion bypass. The length of TLS must be long enough for effective bypass, but it must also be regulated to minimize replication errors by the TLS Pol. The exact position where the TLS Pol ends and the HiFi Pol resumes (i.e. the length of the TLS patch) has not been described. We use steady-state and pre-steady-state kinetic assays to characterize lesion bypass intermediates formed by different archaeal polymerase holoenzyme complexes that include PCNA123 and RFC. After bypass of 8-oxoG by TLS PolY, products accumulate at the template position three base pairs beyond the lesion. PolY is catalytically poor for subsequent extension from this +3 position beyond 8-oxoG, but this inefficiency is overcome by rapid extension of HiFi PolB1. The reciprocation of Pol activities at this intermediate indicates a defined position where TLS Pol extension is limited and where the DNA substrate is handed back to the HiFi Pol after bypass of 8-oxoG.


Assuntos
Proteínas Arqueais/metabolismo , Reparo do DNA , Replicação do DNA , DNA Arqueal/química , DNA Polimerase Dirigida por DNA/metabolismo , Archaea/enzimologia , Archaea/genética , Dano ao DNA , Guanina/análogos & derivados , Guanina/metabolismo
15.
Nucleic Acids Res ; 48(17): 9660-9680, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32890403

RESUMO

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.


Assuntos
Variação Antigênica , DNA Polimerase Dirigida por DNA/metabolismo , Telômero/genética , Trypanosoma brucei brucei/genética , Linhagem Celular , Núcleo Celular/enzimologia , Núcleo Celular/genética , Segregação de Cromossomos , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Regulação da Expressão Gênica , Genoma de Protozoário , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNA , Telômero/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/patogenicidade , Glicoproteínas Variantes de Superfície de Trypanosoma/genética
16.
Proc Natl Acad Sci U S A ; 117(37): 22900-22909, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32873648

RESUMO

Interhomolog recombination (IHR) occurs spontaneously in somatic human cells at frequencies that are low but sufficient to ameliorate some genetic diseases caused by heterozygous mutations or autosomal dominant mutations. Here we demonstrate that DNA nicks or double-strand breaks (DSBs) targeted by CRISPR-Cas9 to both homologs can stimulate IHR and associated copy-neutral loss of heterozygosity (cnLOH) in human cells. The frequency of IHR is 10-fold lower at nicks than at DSBs, but cnLOH is evident in a greater fraction of recombinants. IHR at DSBs occurs predominantly via reciprocal end joining. At DSBs, depletion of POLQ caused a dramatic increase in IHR and in the fraction of recombinants exhibiting cnLOH, suggesting that POLQ promotes end joining in cis, which limits breaks available for recombination in trans These results define conditions that may produce cnLOH as a mutagenic signature in cancer and may, conversely, promote therapeutic correction of both compound heterozygous and dominant negative mutations associated with genetic disease.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA/metabolismo , Reparo de DNA por Recombinação , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Simples , Reparo do DNA por Junção de Extremidades , DNA Ligases/genética , DNA Ligases/metabolismo , DNA Polimerase Dirigida por DNA/genética , Heterozigoto , Humanos , Perda de Heterozigosidade , Mutação , Recombinação Genética
17.
Methods Mol Biol ; 2203: 147-165, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833211

RESUMO

We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA that has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA-dependent RNA polymerase.


Assuntos
Vírus da Bronquite Infecciosa/genética , Transfecção/métodos , Vírus Vaccinia/genética , Animais , Bacteriófagos/genética , Chlorocebus aethiops , DNA Polimerase Dirigida por DNA/metabolismo , Vírus da Varíola das Aves Domésticas/genética , Recombinação Homóloga , Microrganismos Geneticamente Modificados , Vírus Vaccinia/isolamento & purificação , Células Vero
18.
Nat Commun ; 11(1): 4196, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826907

RESUMO

Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. Prim-Pol's function in genome maintenance pathways including replication, repair and damage tolerance. Mycobacteria contain multiple Prim-Pols required for lesion repair, including Prim-PolC that performs short gap repair synthesis during excision repair. To understand the molecular basis of Prim-PolC's gap recognition and synthesis activities, we elucidated crystal structures of pre- and post-catalytic complexes bound to gapped DNA substrates. These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). This mechanism enables Prim-PolC to couple primer extension with template base dislocation, ensuring that the unpaired templating bases in the gap are ushered into the active site in an ordered manner. Insights provided by these structures establishes the molecular basis of Prim-PolC's gap recognition and extension activities, while also illuminating the mechanisms of primer extension utilised by closely related Prim-Pols.


Assuntos
Proteínas de Bactérias/química , DNA Primase/química , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA/química , Mycobacterium/genética , Mycobacterium/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
19.
Nucleic Acids Res ; 48(14): 7844-7855, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652013

RESUMO

The catalytic activity of human AURORA-A kinase (AURKA) regulates mitotic progression, and its frequent overexpression in major forms of epithelial cancer is associated with aneuploidy and carcinogenesis. Here, we report an unexpected, kinase-independent function for AURKA in DNA replication initiation whose inhibition through a class of allosteric inhibitors opens avenues for cancer therapy. We show that genetic depletion of AURKA, or its inhibition by allosteric but not catalytic inhibitors, blocks the G1-S cell cycle transition. A catalytically inactive AURKA mutant suffices to overcome this block. We identify a multiprotein complex between AURKA and the replisome components MCM7, WDHD1 and POLD1 formed during G1, and demonstrate that allosteric but not catalytic inhibitors prevent the chromatin assembly of functional replisomes. Indeed, allosteric but not catalytic AURKA inhibitors sensitize cancer cells to inhibition of the CDC7 kinase subunit of the replication-initiating factor DDK. Thus, our findings define a mechanism essential for replisome assembly during DNA replication initiation that is vulnerable to inhibition as combination therapy in cancer.


Assuntos
Aurora Quinase A/fisiologia , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Regulação Alostérica , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular , Pontos de Checagem da Fase G1 do Ciclo Celular , Células HeLa , Humanos , Interfase/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Origem de Replicação
20.
Nat Commun ; 11(1): 3664, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694532

RESUMO

Ethanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes.


Assuntos
Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Etanol/toxicidade , Taxa de Mutação , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/genética , Mutagênese , Testes de Mutagenicidade , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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