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1.
F1000Res ; 82019.
Artigo em Inglês | MEDLINE | ID: mdl-31602296

RESUMO

DNA topoisomerases are enzymes that catalyze changes in the torsional and flexural strain of DNA molecules. Earlier studies implicated these enzymes in a variety of processes in both prokaryotes and eukaryotes, including DNA replication, transcription, recombination, and chromosome segregation. Studies performed over the past 3 years have provided new insight into the roles of various topoisomerases in maintaining eukaryotic chromosome structure and facilitating the decatenation of daughter chromosomes at cell division. In addition, recent studies have demonstrated that the incorporation of ribonucleotides into DNA results in trapping of topoisomerase I (TOP1)-DNA covalent complexes during aborted ribonucleotide removal. Importantly, such trapped TOP1-DNA covalent complexes, formed either during ribonucleotide removal or as a consequence of drug action, activate several repair processes, including processes involving the recently described nuclear proteases SPARTAN and GCNA-1. A variety of new TOP1 inhibitors and formulations, including antibody-drug conjugates and PEGylated complexes, exert their anticancer effects by also trapping these TOP1-DNA covalent complexes. Here we review recent developments and identify further questions raised by these new findings.


Assuntos
DNA Topoisomerases/fisiologia , Neoplasias , DNA , Dano ao DNA , Replicação do DNA , Humanos
2.
Enzymes ; 45: 1-26, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627875

RESUMO

DNA polymerase ß plays a central role in the base excision DNA repair pathway that cleanses the genome of apurinic/apyrimidinic (AP) sites. AP sites arise in DNA from spontaneous base loss and DNA damage-specific glycosylases that hydrolyze the N-glycosidic bond between the deoxyribose and damaged base. AP sites are deleterious lesions because they can be mutagenic and/or cytotoxic. DNA polymerase ß contributes two enzymatic activities, DNA synthesis and lyase, during the repair of AP sites; these activities reside on carboxyl- and amino-terminal domains, respectively. Accordingly, its cellular, structural, and kinetic attributes have been extensively characterized and it serves as model enzyme for the nucleotidyl transferase reaction utilized by other replicative, repair, and trans-lesion DNA polymerases.


Assuntos
DNA Polimerase beta/metabolismo , Reparo do DNA , Replicação do DNA , Animais , Dano ao DNA , DNA Glicosilases/metabolismo
3.
Enzymes ; 45: 139-181, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627876

RESUMO

DNA contains information that must be safeguarded, but also accessed for transcription and replication. To perform replication, eukaryotic cells use the B-family DNA polymerase enzymes Polδ and Polɛ, which are optimized for accuracy, speed, and processivity. The molecular basis of these high-performance characteristics causes these replicative polymerases to fail at sites of DNA damage (lesions), which would lead to genomic instability and cell death. To avoid this, cells possess additional DNA polymerases such as the Y-family of polymerases and the B-family member Polζ that can replicate over sites of DNA damage in a process called translesion synthesis (TLS). While able to replicate over DNA lesions, the TLS polymerases exhibit low-fidelity on undamaged DNA and, consequently, must be prevented from replicating DNA under normal circumstances and recruited only when necessary. The replicative bypass of most types of DNA lesions requires the consecutive action of these specialized TLS polymerases assembled into a dynamic multiprotein complex called the Rev1/Polζ mutasome. To this end, posttranslational modifications and a network of protein-protein interactions mediated by accessory domains/subunits of the TLS polymerases control the assembly and rearrangements of the Rev1/Polζ mutasome and recruitment of TLS proteins to sites of DNA damage. This chapter focuses on the structures and interactions that control these processes underlying the function of the Rev1/Polζ mutasome, as well as the development of small molecule inhibitors of the Rev1/Polζ-dependent TLS holding promise as a potential anticancer therapy.


Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética
4.
Enzymes ; 45: 183-223, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627877

RESUMO

Hexameric DNA helicases involved in the separation of duplex DNA at the replication fork have a universal architecture but have evolved from two separate protein families. The consequences are that the regulation, translocation polarity, strand specificity, and architectural orientation varies between phage/bacteria to that of archaea/eukaryotes. Once assembled and activated for single strand DNA translocation and unwinding, the DNA polymerase couples tightly to the helicase forming a robust replisome complex. However, this helicase-polymerase interaction can be challenged by various forms of endogenous or exogenous agents that can stall the entire replisome or decouple DNA unwinding from synthesis. The consequences of decoupling can be severe, leading to a build-up of ssDNA requiring various pathways for replication fork restart. All told, the hexameric helicase sits prominently at the front of the replisome constantly responding to a variety of obstacles that require transient unwinding/reannealing, traversal of more stable blocks, and alternations in DNA unwinding speed that regulate replisome progression.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , Complexos Multienzimáticos/metabolismo , DNA/metabolismo , DNA de Cadeia Simples/química
5.
Enzymes ; 45: 289-310, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627881

RESUMO

PrimPol is the second primase discovered in eukaryotic cells, whose function is to restart the stalled replication forks during both mitochondrial and nuclear DNA replication. This chapter revises our current knowledge about the mechanism of synthesis of DNA primers by human PrimPol, and the importance of its distinctive Zn-finger domain (ZnFD). After PrimPol forms a binary complex with ssDNA, the formation of the pre-ternary complex strictly requires the presence of Mn2+ ions to stabilize the interaction of the incoming deoxynucleotide at the 3'-site. The capacity to bind both ssDNA template and 3'-deoxynucleotide was shown to reside in the AEP core of PrimPol, with ZnFD being dispensable at these two early steps of the primase reaction. Sugar selection favoring dNTPs versus NTPs at the 3' site is mediated by a specific tyrosine (Tyr100) acting as a steric gate. Besides, a specific glutamate residue (Glu116) conforming a singular A motif (DxE) promotes the use of Mn2+ to stabilize the pre-ternary complex. Mirroring the function of the PriL subunit of dimeric AEP primases, the ZnFD of PrimPol is crucial to stabilize the initiating 5'-nucleotide, specifically interacting with the gamma-phosphate. Such an interaction is crucial to optimize dimer formation and the subsequent translocation events leading to the processive synthesis of a mature DNA primer. Finally, the capacity of PrimPol to tolerate lesions is discussed in the context of its DNA primase function, and its potential as a TLS primase.


Assuntos
DNA Primase/metabolismo , Primers do DNA/biossíntese , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Humanos
6.
Enzymes ; 45: 311-341, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627882

RESUMO

Mitochondria play a central role in bioenergetics, and fulfill a plethora of functions in cell signaling, programmed cell death, and biosynthesis of key protein cofactors. Mitochondria harbor their own genomic DNA, which encodes protein subunits of the electron transport chain and a full set of transfer and ribosomal RNAs. Mitochondrial DNA (mtDNA) is essential for cellular and organismal functions, and defects in mitochondrial genome maintenance have been implicated in common human diseases and mitochondrial disorders. mtDNA repair and degradation are known pathways to cope with mtDNA damage; however, molecular factors involved in this process have remained unclear. Such knowledge is fundamental to the understanding of mitochondrial genomic maintenance and pathology, because mtDNA degradation may contribute to the etiology of mtDNA depletion syndromes and to the activation of the innate immune response by fragmented mtDNA. This article reviews the current literature regarding the importance of mitochondrial DNA degradation in mtDNA maintenance and stress response, and the recent progress in uncovering molecular factors involved in mtDNA degradation. These factors include key components of the mtDNA replication machinery, such as DNA polymerase γ, helicase Twinkle, and exonuclease MGME1, as well as a major DNA-packaging protein, mitochondrial transcription factor A (TFAM).


Assuntos
DNA Mitocondrial/metabolismo , Genoma Mitocondrial/genética , Estresse Fisiológico , Replicação do DNA , Humanos , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia
7.
Enzymes ; 45: 59-97, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627883

RESUMO

The nucleotide excision repair (NER) system removes a variety of types of helix-distorting lesions from DNA through a dual incision mechanism, in which the damaged nucleotide bases are excised in the form of a small, excised, damage-containing single-stranded DNA oligonucleotide (sedDNA). Damage removal leaves a gap in the DNA template that must then be filled in by the action of a DNA polymerase and ligated to the downstream phosphodiester backbone in the DNA to complete the repair reaction. Defects in damage removal, sedDNA processing, or gap filling have the potential to be mutagenic and lethal to cells, and thus several human pathologies, including cancer and aging, are associated with defects in NER. This review summarizes our current understanding of NER with a focus on the enzymes that excise sedDNAs and restore the duplex DNA to its native state in human cells.


Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA/química , DNA/metabolismo , DNA/biossíntese , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos
8.
Postepy Biochem ; 65(2): 143-152, 2019 06 06.
Artigo em Polonês | MEDLINE | ID: mdl-31642653

RESUMO

High replication fidelity, understood as the DNA polymerases' ability to select nucleotides with both correct base and sugar, is of critical importance for maintaining the genetic stability. Due to the fact that the cellular levels of ribonucleotides are much higher than the concentrations of deoxyribonucleotides, replicative polymerases are able to incorporate ribonucleotides with up to 1000-fold higher frequency than mismatched deoxyribonucleotides. The ability to discriminate against ribonucleotides by the DNA polymerases relies on the steric gate residue in the enzyme's catalytic centre. Despite the fact that ribonucleotides are the most abundantly inserted incorrect nucleotides in DNA, they are not observed in properly functioning cells. The major pathway responsible for the recognition and removal of ribonucleotides from DNA is called Ribonucleotide Excision Repair. The impairment of ribonucleotide removal pathways can cause increased mutation rate, replication stress, DNA breakage, problems with transcription, chromatin structure maintenance, genetic disorders and cell death. In spite of that, ribonucleotide incorporation into DNA may have some positive biological impact, stimulating mismatch repair and non-homologous end joining.


Assuntos
Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/química , DNA/metabolismo , Instabilidade Genômica , Ribonucleotídeos/metabolismo , DNA/genética , Replicação do DNA , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Ribonucleotídeos/química
9.
Postepy Biochem ; 65(3): 202-211, 2019 10 01.
Artigo em Polonês | MEDLINE | ID: mdl-31643167

RESUMO

Advances in high resolution microscopy techniques and development of high throughput DNA analyses allow to reconsider the views concerning bacterial chromosome (nucleoid). Recent reports show that nucleoid exhibits a hierarchical organization, similarly to the eukaryotic chromatin. However, bacterial chromosome undergoes constant modifications and topological rearrangements due to the ongoing DNA replication, transcription and translation processes. Organization of dynamic and highly compacted nucleoid structure depends on physical factors acting on chromosome molecule inside small cell compartment, and is a consequence of action of many different DNA-binding proteins. The main goal of this review is to present the recent reports on bacterial chromatin structure and to elucidate the physical and molecular factors influencing its intracellular organization.


Assuntos
Bactérias/genética , Cromatina/metabolismo , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Bactérias/metabolismo , Cromatina/química , Cromatina/genética , Cromossomos Bacterianos/química , Cromossomos Bacterianos/genética , Replicação do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo
10.
Cytogenet Genome Res ; 159(1): 48-53, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31610539

RESUMO

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.


Assuntos
Amaryllidaceae/genética , Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Replicação do DNA/genética , Zea mays/genética , Cromatina/metabolismo , Cromossomos/genética , DNA de Plantas/genética , Endonucleases/genética , Hibridização in Situ Fluorescente/métodos , RNA Guia/genética , Telômero/genética
11.
Nucleic Acids Res ; 47(18): 9495-9501, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31504784

RESUMO

We document the preparation and properties of dimerized pentaphosphate-bridged deoxynucleotides (dicaptides) that contain reactive components of two different nucleotides simultaneously. Importantly, dicaptides are found to be considerably more stable to hydrolysis than standard dNTPs. Steady-state kinetics studies show that the dimers exhibit reasonably good efficiency with the Klenow fragment of DNA polymerase I, and we identify thermostable enzymes that process them efficiently at high temperature. Experiments show that the dAp5dT dimer successfully acts as a combination of dATP and dTTP in primer extension reactions, and the dGp5dC dimer as a combination of dGTP and dCTP. The two dimers in combination promote successful 4-base primer extension. The final byproduct of the reaction, triphosphate, is shown to be less inhibitory to primer extension than pyrophosphate, the canonical byproduct. Finally, we document PCR amplification of DNA with two dimeric nucleotides, and show that the dimers can promote amplification under extended conditions when PCR with normal dNTPs fails. These dimeric nucleotides represent a novel and simple approach for increasing stability of nucleotides and avoiding inhibition from pyrophosphate.


Assuntos
DNA Polimerase I/genética , Replicação do DNA/genética , DNA/biossíntese , Nucleotídeos/genética , DNA/genética , Nucleotídeos de Desoxicitosina/genética , Nucleotídeos de Desoxiguanina/genética , Cinética , Temperatura Ambiente
12.
Zhongguo Dang Dai Er Ke Za Zhi ; 21(9): 876-880, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31506145

RESUMO

OBJECTIVE: To study the correlation of Mycoplasma pneumoniae DNA (MP-DNA) replication level in throat swab and bronchoalveolar lavage fluid (BALF) with disease severity in children with severe Mycoplasma pneumoniae pneumonia (SMPP). METHODS: A total of 44 children with SMPP who underwent bronchoalveolar lavage were enrolled as subjects. The serum levels of cytokines and MP-DNA replication times in throat swab were measured in the acute stage and the recovery stage, and the levels of interleukin (IL)-8 and MP-DNA replication times in BALF were measured in the acute stage. According to whether mechanical ventilation was needed for respiratory failure, the children were divided into a mechanical ventilation group (n=19) and a non-mechanical ventilation group (n=25), and the two groups were compared in MP-DNA replication times in BALF. RESULTS: For the children with SMPP, serum levels of C-reactive protein, erythrocyte sedimentation rate, lactate dehydrogenase, IL-1, IL-6, IL-8, and IL-18 in the acute stage were significantly higher than those in the recovery stage (P<0.05). In the acute stage, MP-DNA replication times in throat swab were positively correlated with those in BALF (r=0.613, P<0.05), and MP-DNA replication times in BALF were positively correlated with IL-18 levels in peripheral blood and BALF (r=0.613 and 0.41 respectively, P<0.05). Compared with the non-mechanical ventilation group, the mechanical ventilation group had significantly higher MP-DNA replication times in BALF, a significantly longer duration of systemic hormone treatment, significantly higher serum levels of lactate dehydrogenase and IL-18, and significantly higher white blood cell count and IL-18 level in BALF (P<0.05). CONCLUSIONS: In children with SMPP, MP-DNA replication level in throat swab and BALF can be used as a reference index for the assessment of disease severity.


Assuntos
Mycoplasma pneumoniae , Pneumonia por Mycoplasma , Líquido da Lavagem Broncoalveolar , Criança , Citocinas , Replicação do DNA , DNA Bacteriano , Humanos
13.
Mol Biol (Mosk) ; 53(4): 600-612, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397434

RESUMO

A new plasmid, pSM22, was isolated from Serratia marcescens and sequenced. Its 43 190-bp sequence with an average GC-content of 58% contains 31 open reading frames (ORFs) which form replication, conjugation, stability, and adaptive modules. The replication module includes a site of initiation of leading-strand synthesis in plasmid replication, a replication termination site (terC), the rep A (=repA1) and repA4 genes, and the copA sequence, which codes for an antisense RNA (asRNA). These structures are functionally integrated in an FII replicon (incompatibility group IncFII). Based on the significant differences between the FII replicon and the canonical sequences of the R plasmids R1 and NR1 (=R100=R222), pSM22 was assigned to a new subtype. The conjugation module includes 13 genes with a high identity to the genes responsible for conjugation of the F plasmid. A comparative genomic analysis showed that the conjugation modules of pSM22 and F are structurally similar. By the conjugation system and the presence of three conserved motifs in relaxase (TraI), pSM22 belongs to the F12 clade of the MOBF type. The stability module includes the resD and parA genes, which are responsible for the resolution of multimeric plasmid forms and their subsequent segregation between daughter cells. The adaptive module contains the microcin H47 (MccH47) secretion/processing and UV resistance genes. The mosaic structure of pSM22 and reductive evolution of its modules suggest high genomic plasticity for the genus Serratia. An analysis of the architecture of the pSM22 modules clarifies the evolutionary relationships among IncF/MOBF12 group plasmids in bacteria of the family Enterobacteriaceae and opens a novel avenue for further comparative genomic studies of Serratia plasmids.


Assuntos
Evolução Molecular , Genômica , Plasmídeos/classificação , Plasmídeos/genética , Replicação do DNA , Genes Bacterianos , Genoma Bacteriano/genética , Replicon/genética , Serratia marcescens/genética
14.
Nucleic Acids Res ; 47(18): 9666-9684, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31392335

RESUMO

Break induced replication (BIR) is a double strand break repair pathway that can promote genetic instabilities similar to those observed in cancer. Instead of a replication fork, BIR is driven by a migration bubble where asynchronous synthesis between leading and lagging strands leads to accumulation of single-stranded DNA (ssDNA) that promotes mutation. However, the details of the mechanism of mutagenesis, including the identity of the participating proteins, remain unknown. Using yeast as a model, we demonstrate that mutagenic ssDNA is formed at multiple positions along the BIR track and that Pol ζ is responsible for the majority of both spontaneous and damage-induced base substitutions during BIR. We also report that BIR creates a potent substrate for APOBEC3A (A3A) cytidine deaminase that can promote formation of mutation clusters along the entire track of BIR. Finally, we demonstrate that uracil glycosylase initiates the bypass of DNA damage induced by A3A in the context of BIR without formation of base substitutions, but instead this pathway frequently leads to chromosomal rearrangements. Together, the expression of A3A during BIR in yeast recapitulates the main features of APOBEC-induced kataegis in human cancers, suggesting that BIR might represent an important source of these hyper-mutagenic events.


Assuntos
Cromossomos/genética , Citidina Desaminase/genética , Reparo do DNA/genética , Proteínas/genética , Recombinação Genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Humanos , Mutagênese/genética , Mutação , Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
15.
Nucleic Acids Res ; 47(18): 9685-9695, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31410468

RESUMO

Common fragile sites (CFSs) are genomic regions prone to breakage under replication stress conditions recurrently rearranged in cancer. Many CFSs are enriched with AT-dinucleotide rich sequences (AT-DRSs) which have the potential to form stable secondary structures upon unwinding the double helix during DNA replication. These stable structures can potentially perturb DNA replication progression, leading to genomic instability. Using site-specific targeting system, we show that targeted integration of a 3.4 kb AT-DRS derived from the human CFS FRA16C into a chromosomally stable region within the human genome is able to drive fragile site formation under conditions of replication stress. Analysis of >1300 X chromosomes integrated with the 3.4 kb AT-DRS revealed recurrent gaps and breaks at the integration site. DNA sequences derived from the integrated AT-DRS showed in vitro a significantly increased tendency to fold into branched secondary structures, supporting the predicted mechanism of instability. Our findings clearly indicate that intrinsic DNA features, such as complexed repeated sequence motifs, predispose the human genome to chromosomal instability.


Assuntos
Instabilidade Cromossômica/genética , Sítios Frágeis do Cromossomo/genética , DNA/genética , Repetições de Dinucleotídeos/genética , Replicação do DNA/genética , Genoma Humano , Humanos , Conformação de Ácido Nucleico
16.
Nat Cell Biol ; 21(8): 952-965, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31358968

RESUMO

The differential distribution of the microtubule-organizing centres (MTOCs) that orchestrate spindle formation during cell division is a fascinating phenomenon originally described in Saccharomyces cerevisiae and later found to be conserved during stem cell divisions in organisms ranging from Drosophila to humans. Whether predetermined MTOC inheritance patterns fulfil any biological function is however unknown. Using a genetically designed S. cerevisiae strain that displays a constitutively inverted MTOC fate, we demonstrate that the asymmetric segregation of these structures is critical to ensure normal levels of the Sir2 sirtuin and correct localization of the mitochondrial inheritance regulator Mfb1, and therefore to properly distribute functional mitochondria and protein aggregates between the mother and daughter cells. Consequently, interfering with this process severely accelerates cellular ageing.


Assuntos
Longevidade/fisiologia , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Replicação do DNA/fisiologia , Proteínas F-Box/metabolismo , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo
17.
Microbes Environ ; 34(3): 316-326, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31353332

RESUMO

Thermococcus kodakarensis possesses two DNA polymerases, Pol B and Pol D. We generated a T. kodakarensis strain (DPB1) in which polB was completely deleted and a derivative of DPB1 in which polB was overexpressed; neither of the generated strains exhibited any growth delay, indicating that the lack or overexpression of Pol B in T. kodakarensis did not affect cell growth. We also found that DPB1 showed higher sensitivity to four DNA-damaging agents (ultraviolet C irradiation, γ-ray irradiation, methyl methanesulfonate, and mitomycin C) than the parental strain. The sensitivity of DPB1 was restored to the level of the parent strain by the introduction of a plasmid harboring polB, suggesting that the DNA damage-sensitive phenotype of DPB1 was due to the loss of polB. Collectively, these results indicate that Pol B is involved in DNA repair, but not DNA replication, which, in turn, implies that Pol D is the sole replicative DNA polymerase in Thermococcus species.


Assuntos
Reparo do DNA/genética , DNA Arqueal/genética , DNA Polimerase Dirigida por DNA/genética , Thermococcus/enzimologia , Thermococcus/genética , Proteínas de Bactérias/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Replicação do DNA , Deleção de Genes , Expressão Gênica , Inibidores da Síntese de Ácido Nucleico/farmacologia , Thermococcus/efeitos dos fármacos
18.
J Microbiol Biotechnol ; 29(8): 1273-1280, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31337186

RESUMO

Edwardsiella piscicida is the causative agent of edwardsiellosis, which has caused enormous economic losses worldwide. In our previous research, an attenuated live vaccine WED based on the virulent strain E. piscicida EIB202 can effectively protect turbots against edwardsiellosis via intraperitoneal injection, while vaccination by immersion exhibits a weaker effect. During the development of the immersion vaccine, we surprisingly found the counts of ΔpEIB202/ EIB202 colonized on zebrafish were 100 times lower than those of EIB202. However, pEIB202 carries 53 predicted ORFs and has several copies in E. piscicida EIB202, impeding the study of its function. Thus the replication region is located at a 1 980 bp fragment (from 18 837 to 20 816 bp), containing a transcriptional repressor and a replication protein. Moreover, the minimal replication plasmid, named pRep-q77, has low copies in both E. coli and E. piscicida, but is more stable in E. piscicida than in E. coli. This work lays a foundation for further examination of the function of the virulence plasmid pEIB202.


Assuntos
Replicação do DNA , Edwardsiella/genética , Plasmídeos/genética , Plasmídeos/isolamento & purificação , Animais , Proteínas de Bactérias/genética , DNA Bacteriano/isolamento & purificação , Edwardsiella/crescimento & desenvolvimento , Infecções por Enterobacteriaceae , Escherichia coli/genética , Doenças dos Peixes/microbiologia , Brânquias/microbiologia , Análise de Sequência de DNA , Pele/microbiologia , Virulência/genética , Peixe-Zebra/microbiologia
19.
Nucleic Acids Res ; 47(16): 8595-8605, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31340040

RESUMO

G-quadruplexes (G4s) are stable secondary structures that can lead to the stalling of replication forks and cause genomic instability. Pif1 is a 5' to 3' helicase, localized to both the mitochondria and nucleus that can unwind G4s in vitro and prevent fork stalling at G4 forming sequences in vivo. Using in vitro primer extension assays, we show that both G4s and stable hairpins form barriers to nuclear and mitochondrial DNA polymerases δ and γ, respectively. However, while single-stranded DNA binding proteins (SSBs) readily promote replication through hairpins, SSBs are only effective in promoting replication through weak G4s. Using a series of G4s with increasing stabilities, we reveal a threshold above which G4 through-replication is inhibited even with SSBs present, and Pif1 helicase is required. Because Pif1 moves along the template strand with a 5'-3'-directionality, head-on collisions between Pif1 and polymerase δ or γ result in the stimulation of their 3'-exonuclease activity. Both nuclear RPA and mitochondrial SSB play a protective role during DNA replication by preventing excessive DNA degradation caused by the helicase-polymerase conflict.


Assuntos
DNA Helicases/genética , DNA Polimerase III/genética , Polimerase do DNA Mitocondrial/genética , DNA Fúngico/genética , Quadruplex G , Proteína de Replicação A/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Núcleo Celular/metabolismo , DNA Helicases/metabolismo , DNA Polimerase III/metabolismo , Polimerase do DNA Mitocondrial/metabolismo , Replicação do DNA , DNA Fúngico/química , DNA Fúngico/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Genoma Fúngico , Instabilidade Genômica , Mitocôndrias/metabolismo , Ligação Proteica , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Anticancer Res ; 39(7): 3565-3570, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262880

RESUMO

BACKGROUND/AIM: Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil that has been approved for the treatment of metastatic colorectal cancer. In this study, a comprehensive analysis of DNA replication profile in FTD-treated colon cancer cells was performed. MATERIALS AND METHODS: HCT-116 cells were exposed to BrdU or FTD and subjected to DNA immunoprecipitation. Immunoprecipitated DNA was sequenced; the density of aligned reads along the genome was calculated. Peak finding, gene ontology, and motif analysis were performed using MACS, GREAT, and MEME, respectively. RESULTS: We identified 6,043 and 5,080 high-confidence FTD and BrdU peaks in HCT-116 cells, respectively. Of 6,043 FTD peaks, 2,911 peaks were uncommon to BrdU. We observed that FTD was preferentially incorporated into genomic regions containing simple repeats, CpG islands, and gene bodies. Conserved motifs in FTD peaks contained dinucleotide repeats such as (GT)n. CONCLUSION: Global FTD incorporation patterns delineated FTD, preferentially incorporating loci in cancer cells.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Bromodesoxiuridina/farmacologia , Neoplasias Colorretais/genética , Replicação do DNA/efeitos dos fármacos , Trifluridina/farmacologia , Células HCT116 , Humanos , Análise de Sequência de DNA
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