Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.855
Filtrar
1.
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
2.
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
3.
Mol Cell ; 79(6): 1037-1050.e5, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32882183

RESUMO

DNA double-stranded breaks (DSBs) are dangerous lesions threatening genomic stability. Fidelity of DSB repair is best achieved by recombination with a homologous template sequence. In yeast, transcript RNA was shown to template DSB repair of DNA. However, molecular pathways of RNA-driven repair processes remain obscure. Utilizing assays of RNA-DNA recombination with and without an induced DSB in yeast DNA, we characterize three forms of RNA-mediated genomic modifications: RNA- and cDNA-templated DSB repair (R-TDR and c-TDR) using an RNA transcript or a DNA copy of the RNA transcript for DSB repair, respectively, and a new mechanism of RNA-templated DNA modification (R-TDM) induced by spontaneous or mutagen-induced breaks. While c-TDR requires reverse transcriptase, translesion DNA polymerase ζ (Pol ζ) plays a major role in R-TDR, and it is essential for R-TDM. This study characterizes mechanisms of RNA-DNA recombination, uncovering a role of Pol ζ in transferring genetic information from transcript RNA to DNA.


Assuntos
DNA/genética , RNA/genética , Saccharomyces cerevisiae/genética , Adolescente , Adulto , DNA/ultraestrutura , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Replicação do DNA/genética , DNA Complementar/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/ultraestrutura , Instabilidade Genômica/genética , Humanos , Pessoa de Meia-Idade , RNA/ultraestrutura , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Adulto Jovem
4.
BMC Med Genet ; 21(1): 167, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32838755

RESUMO

BACKGROUND: Mutations in the exonuclease domain of POLE, a DNA polymerase associated with DNA replication and repair, lead to cancers with ultra-high mutation rates. Most studies focus on intestinal and uterine cancers with POLE mutations. These cancers exhibit a significant immune cell infiltrate and favorable prognosis. We questioned whether loss of function of other DNA polymerases can cooperate to POLE to generate the ultramutator phenotype. METHODS: We used cases and data from 15 cancer types in The Cancer Genome Atlas to investigate mutation frequencies of 14 different DNA polymerases. We tested whether tumor mutation burden, patient outcome (disease-free survival) and immune cell infiltration measured by ESTIMATE can be attributed to mutations in POLQ and POLZ/REV3L. RESULTS: Thirty six percent of colorectal, stomach and endometrial cancers with POLE mutations carried additional mutations in POLQ (E/Q), POLZ/REV3L (E/Z) or both DNA polymerases (E/Z/Q). The mutation burden in these tumors was significantly greater compared to POLE-only (E) mutant tumors (p < 0.001). In addition, E/Q, E/Z, and E/Q/Z mutant tumors possessed an increased frequency of mutations in the POLE exonuclease domain (p = 0.013). Colorectal, stomach and endometrial E/Q, E/Z, and E/Q/Z mutant tumors within TCGA demonstrated 100% disease-free survival, even if the POLE mutations occurred outside the exonuclease domain (p = 0.003). However, immune scores in these tumors were related to microsatellite instability (MSI) and not POLE mutation status. This suggests that the host immune response may not be the sole mechanism for prolonged disease-free survival of ultramutated tumors in this cohort. CONCLUSION: Results in this study demonstrate that mutations in POLQ and REV3L in POLE mutant tumors should undergo further investigation to determine whether POLQ and REV3L mutations contribute to the ultramutator phenotype and favorable outcome of patients with POLE mutant tumors.


Assuntos
Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Exonucleases/genética , Mutação , Neoplasias/genética , Estudos de Coortes , Exonucleases/química , Exonucleases/metabolismo , Feminino , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/metabolismo , Estimativa de Kaplan-Meier , Masculino , Instabilidade de Microssatélites , Neoplasias/classificação , Neoplasias/enzimologia , Domínios Proteicos , Sequenciamento Completo do Exoma/estatística & dados numéricos
5.
Nat Commun ; 11(1): 3615, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32680986

RESUMO

Failure to preserve the integrity of the genome is a hallmark of cancer. Recent studies have revealed that loss of the capacity to repair DNA breaks via homologous recombination (HR) results in a mutational profile termed BRCAness. The enzymatic activity that repairs HR substrates in BRCA-deficient conditions to produce this profile is currently unknown. We here show that the mutational landscape of BRCA1 deficiency in C. elegans closely resembles that of BRCA1-deficient tumours. We identify polymerase theta-mediated end-joining (TMEJ) to be responsible: knocking out polq-1 suppresses the accumulation of deletions and tandem duplications in brc-1 and brd-1 animals. We find no additional back-up repair in HR and TMEJ compromised animals; non-homologous end-joining does not affect BRCAness. The notion that TMEJ acts as an alternative to HR, promoting the genome alteration of HR-deficient cells, supports the idea that polymerase theta is a promising therapeutic target for HR-deficient tumours.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Reparo do DNA por Junção de Extremidades , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Animais , Animais Geneticamente Modificados , Análise Mutacional de DNA , DNA Polimerase Dirigida por DNA/genética , Técnicas de Inativação de Genes , Mutação
6.
Nucleic Acids Res ; 48(15): 8461-8473, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32633759

RESUMO

DNA polymerase ζ (Pol ζ) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases η, ζ and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based interstrand crosslink (ICL) with an 8-atom linker between the crosslinked bases. The Rev1-Pol ζ complex was most efficient in complete bypass synthesis, by 2-3 fold, compared to Pol ζ alone or Pol η. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol η, Pol ζ, and Rev1-Pol ζ. Rev1-Pol ζ, and particularly Pol ζ alone showed a tendency to stall before the ICL, whereas Pol η stalled just after insertion across the ICL. The stalling of Pol η directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol η providing a barrier to further elongation of the correct primer. No stalling by Rev1-Pol ζ directly past the ICL was observed, suggesting that the proposed function of Pol ζ as an extender DNA polymerase is also required for ICL repair.


Assuntos
DNA Polimerase Dirigida por DNA/genética , DNA/genética , Nucleotidiltransferases/genética , Proteínas de Saccharomyces cerevisiae/genética , Estruturas Cromossômicas/efeitos dos fármacos , Estruturas Cromossômicas/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/genética , Complexos Multiproteicos/genética , Compostos de Mostarda Nitrogenada/farmacologia , Saccharomyces cerevisiae/genética
7.
Nucleic Acids Res ; 48(15): 8591-8600, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32644133

RESUMO

In nature, allostery is the principal approach for regulating cellular processes and pathways. Inspired by nature, structure-switching aptamer-based nanodevices are widely used in artificial biotechnologies. However, the canonical aptamer structures in the nanodevices usually adopt a duplex form, which limits the flexibility and controllability. Here, a new regulating strategy based on a clamp-like triplex aptamer structure (CLTAS) was proposed for switching DNA polymerase activity via conformational changes. It was demonstrated that the polymerase activity could be regulated by either adjusting structure parameters or dynamic reactions including strand displacement or enzymatic digestion. Compared with the duplex aptamer structure, the CLTAS possesses programmability, excellent affinity and high discrimination efficiency. The CLTAS was successfully applied to distinguish single-base mismatches. The strategy expands the application scope of triplex structures and shows potential in biosensing and programmable nanomachines.


Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Técnicas Biossensoriais , DNA Polimerase Dirigida por DNA/genética , Taq Polimerase/genética , Aptâmeros de Nucleotídeos/genética , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/farmacologia , Humanos , Nanoestruturas/química , Conformação de Ácido Nucleico/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Taq Polimerase/antagonistas & inibidores , Taq Polimerase/química
8.
Nucleic Acids Res ; 48(15): e87, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32573728

RESUMO

Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50-200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.


Assuntos
DNA Polimerase Dirigida por DNA/genética , Desoxirribonucleotídeos/isolamento & purificação , Oligonucleotídeos/genética , Animais , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/química , Desoxirribonucleotídeos/genética , Camundongos , Inibidores da Síntese de Ácido Nucleico/química , Oligonucleotídeos/síntese química , Ribonuclease H/genética
9.
Proc Natl Acad Sci U S A ; 117(22): 12368-12374, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32409608

RESUMO

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that establishes life-long infection and increases the risk for the development of several cancers and autoimmune diseases. The mechanisms by which chronic EBV infection leads to subsequent disease remain incompletely understood. Lytic reactivation plays a central role in the development of EBV-driven cancers and may contribute to other EBV-associated diseases. Thus, the clinical use of antivirals as suppressive therapy for EBV lytic reactivation may aid efforts aimed at disease prevention. Current antivirals for EBV have shown limited clinical utility due to low potency or high toxicity, leaving open the need for potent antivirals suitable for long-term prophylaxis. In the present study, we show that tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF), drugs with excellent safety profiles used clinically for HIV prevention, inhibit EBV lytic DNA replication, with respective IC50 values of 0.30 µM and 84 nM. In a cell-based assay, TAF was 35- and 24-fold and TDF was 10- and 7-fold more potent than acyclovir and penciclovir, respectively, and TAF was also twice as potent as ganciclovir. The active metabolite of tenofovir prodrugs, tenofovir-diphosphate, inhibited the incorporation of dATP into a primed DNA template by the EBV DNA polymerase in vitro. In contrast to acyclovir, treatment of cells during latency for 24 h with TAF still inhibited EBV lytic DNA replication at 72 h after drug was removed. Our results suggest that tenofovir prodrugs may be particularly effective as inhibitors of EBV lytic reactivation, and that clinical studies to address critical questions about disease prevention are warranted.


Assuntos
Antivirais/farmacologia , Replicação do DNA/efeitos dos fármacos , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Tenofovir/farmacologia , Proteínas Virais/antagonistas & inibidores , DNA Viral/genética , DNA Viral/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Herpesvirus Humano 4/enzimologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , Pró-Fármacos/farmacologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
10.
Mol Cell ; 79(1): 140-154.e7, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32464091

RESUMO

Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.


Assuntos
DNA Primase/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DnaB Helicases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Holoenzimas/química , DNA Primase/genética , DNA Bacteriano , DNA Polimerase Dirigida por DNA/genética , DnaB Helicases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Holoenzimas/genética , Holoenzimas/metabolismo , Conformação Molecular , Ligação Proteica , Conformação Proteica
11.
Nat Chem Biol ; 16(6): 610-619, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32444838

RESUMO

Continuous directed evolution methods allow the key steps of evolution-gene diversification, selection, and replication-to proceed in the laboratory with minimal researcher intervention. As a result, continuous evolution can find solutions much more quickly than traditional discrete evolution methods. Continuous evolution also enables the exploration of longer and more numerous evolutionary trajectories, increasing the likelihood of accessing solutions that require many steps through sequence space and greatly facilitating the iterative refinement of selection conditions and targeted mutagenesis strategies. Here we review the historical advances that have expanded continuous evolution from its earliest days as an experimental curiosity to its present state as a powerful and surprisingly general strategy for generating tailor-made biomolecules, and discuss more recent improvements with an eye to the future.


Assuntos
Evolução Molecular , Mutagênese , Proteínas/genética , Allolevivirus/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Evolução Molecular Direcionada , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Engenharia de Proteínas
12.
BMC Med Genet ; 21(1): 93, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32375772

RESUMO

BACKGROUND: Pathogenic SLC6A1 variants have been reported in patients with myoclonic-atonic epilepsy (MAE). NOTCH1, encoding a member of the Notch family of proteins, is known to be associated with aortic valve disease. The PRIMPOL variant has only been identified in Chinese patients with high myopia. Exome sequencing analysis now allows the simultaneous detection of multiple genetic etiologies for patients with complicated clinical features. However, the presence of three Mendelian disorders in one patient supported by their respective pathogenic variants and clinical phenotypes is very rare. CASE PRESENTATION: Here, we report a 4-year-old Chinese boy who presented with MAE, delayed language, borderline intellectual disability (ID), mildly impaired social skills and attention deficit hyperactivity disorder (ADHD). He also had mild aortic valve stenosis and high myopia. Using whole-exome sequencing (WES), we identified three variants: (1) SLC6A1, NM_003042.4: c.881-883del (p.Phe294del), (2) NOTCH1, NM_017617.5:c.1100-2A > G and (3) PRIMPOL, NM_152683.4:c.265 T > G (p.Tyr89Asp). Parental Sanger sequencing confirmed that SLC6A1 and NOTCH1 variants were de novo, whereas the PRIMPOL variant was inherited from the father who also had high myopia. Furthermore, the PRIMPOL variant was absent from the genomes of the paternal grandparents, and thus was also a de novo event in the family. All three variants are classified as pathogenic. CONCLUSION: The SLC6A1 variant could explain the features of MAE, delayed language, borderline ID, impaired social skills and ADHD in this patient, whereas the features of aortic valve stenosis and high myopia of the patient may be explained by variants in NOTCH1 and PRIMPOL, respectively. This case demonstrated the utility of exome sequencing in uncovering the multiple pathogenic variants in a patient with complicated phenotypes due to the blending of three Mendelian disorders.


Assuntos
Epilepsias Mioclônicas/genética , Epilepsia Generalizada/genética , Predisposição Genética para Doença , Miopia/genética , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/patologia , Pré-Escolar , DNA Primase/genética , DNA Polimerase Dirigida por DNA/genética , Epilepsias Mioclônicas/patologia , Epilepsia Generalizada/patologia , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Testes Genéticos , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Masculino , Enzimas Multifuncionais/genética , Mutação/genética , Miopia/patologia , Receptor Notch1/genética , Sequenciamento Completo do Exoma
13.
Nucleic Acids Res ; 48(9): 5119-5134, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32282906

RESUMO

Reactive oxygen species generate the genotoxic 8-oxoguanine (oxoG) and 8-oxoadenine (oxoA) as major oxidative lesions. The mutagenicity of oxoG is attributed to the lesion's ability to evade the geometric discrimination of DNA polymerases by adopting Hoogsteen base pairing with adenine in a Watson-Crick-like geometry. Compared with oxoG, the mutagenesis mechanism of oxoA, which preferentially induces A-to-C mutations, is poorly understood. In the absence of protein contacts, oxoA:G forms a wobble conformation, the formation of which is suppressed in the catalytic site of most DNA polymerases. Interestingly, human DNA polymerase η (polη) proficiently incorporates dGTP opposite oxoA, suggesting the nascent oxoA:dGTP overcomes the geometric discrimination of polη. To gain insights into oxoA-mediated mutagenesis, we determined crystal structures of polη bypassing oxoA. When paired with dGTP, oxoA adopted a syn-conformation and formed Hoogsteen pairing while in a wobble geometry, which was stabilized by Gln38-mediated minor groove contacts to oxoA:dGTP. Gln38Ala mutation reduced misinsertion efficiency ∼55-fold, indicating oxoA:dGTP misincorporation was promoted by minor groove interactions. Also, the efficiency of oxoA:dGTP insertion by the X-family polß decreased ∼380-fold when Asn279-mediated minor groove contact to dGTP was abolished. Overall, these results suggest that, unlike oxoG, oxoA-mediated mutagenesis is greatly induced by minor groove interactions.


Assuntos
Adenina/análogos & derivados , DNA Polimerase Dirigida por DNA/química , Mutagênese , Adenina/química , Pareamento de Bases , DNA Polimerase beta/química , DNA Polimerase beta/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxiguanina/química , Nucleotídeos de Desoxiguanina/metabolismo , Humanos , Cinética , Mutação , Nucleotídeos de Timina/metabolismo
14.
Mutat Res ; 852: 503164, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32265042

RESUMO

In central Brazil, in the municipality of Faina (state of Goiás), the small and isolated village of Araras comprises a genetic cluster of xeroderma pigmentosum (XP) patients. The high level of consanguinity and the geographical isolation gave rise to a high frequency of XP patients. Recently, two founder events were identified affecting that community, with two independent mutations at the POLH gene, c.764 + 1 G > A (intron 6) and c.907 C > T; p.Arg303* (exon 8). These deleterious mutations lead to the xeroderma pigmentosum variant syndrome (XP-V). Previous reports identified both mutations in other countries: the intron 6 mutation in six patients (four families) from Northern Spain (Basque Country and Cantabria) and the exon 8 mutation in two patients from different families in Europe, one of them from Kosovo. In order to investigate the ancestry of the XP patients and the age for these mutations at Araras, we generated genotyping information for 22 XP-V patients from Brazil (16), Spain (6) and Kosovo (1). The local genomic ancestry and the shared haplotype segments among the patients showed that the intron 6 mutation at Araras is associated with an Iberian genetic legacy. All patients from Goiás, homozygotes for intron 6 mutation, share with the Spanish patients identical-by-descent (IBD) genomic segments comprising the mutation. The entrance date for the Iberian haplotype at the village was calculated to be approximately 200 years old. This result is in agreement with the historical arrival of Iberian individuals at the Goiás state (BR). Patients from Goiás and the three families from Spain share 1.8 cM (family 14), 1.7 cM (family 15), and a more significant segment of 4.7 cM within family 13. On the other hand, the patients carrying the exon 8 mutation do not share any specific genetic segment, indicating an old genetic distance between them or even no common ancestry.


Assuntos
DNA Polimerase Dirigida por DNA/genética , Haplótipos , Padrões de Herança , Mutação , Isolamento Reprodutivo , Xeroderma Pigmentoso/genética , Brasil/epidemiologia , Consanguinidade , Europa (Continente)/epidemiologia , Éxons , Feminino , Genética Populacional , Heterozigoto , Homozigoto , Migração Humana , Humanos , Íntrons , Masculino , Fenótipo , Xeroderma Pigmentoso/epidemiologia , Xeroderma Pigmentoso/patologia
15.
Proc Natl Acad Sci U S A ; 117(17): 9440-9450, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32277034

RESUMO

Yeast strains with low levels of the replicative DNA polymerases (alpha, delta, and epsilon) have high levels of chromosome deletions, duplications, and translocations. By examining the patterns of mutations induced in strains with low levels of DNA polymerase by the human protein APOBEC3B (a protein that deaminates cytosine in single-stranded DNA), we show dramatically elevated amounts of single-stranded DNA relative to a wild-type strain. During DNA replication, one strand (defined as the leading strand) is replicated processively by DNA polymerase epsilon and the other (the lagging strand) is replicated as short fragments initiated by DNA polymerase alpha and extended by DNA polymerase delta. In the low DNA polymerase alpha and delta strains, the APOBEC-induced mutations are concentrated on the lagging-strand template, whereas in the low DNA polymerase epsilon strain, mutations occur on the leading- and lagging-strand templates with similar frequencies. In addition, for most genes, the transcribed strand is mutagenized more frequently than the nontranscribed strand. Lastly, some of the APOBEC-induced clusters in strains with low levels of DNA polymerase alpha or delta are greater than 10 kb in length.


Assuntos
Citidina Desaminase/farmacologia , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas Fúngicas/metabolismo , Antígenos de Histocompatibilidade Menor/farmacologia , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos , Replicação do DNA , DNA Fúngico , DNA Polimerase Dirigida por DNA/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Estudo de Associação Genômica Ampla , Humanos , Mutação , Análise de Sequência de DNA/métodos
16.
PLoS Genet ; 16(4): e1008759, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32330130

RESUMO

Bases within DNA are frequently damaged, producing obstacles to efficient and accurate DNA replication by replicative polymerases. Translesion synthesis (TLS) polymerases, via their ability to catalyze nucleotide additions to growing DNA chains across DNA lesions, promote replication of damaged DNA, thus preventing checkpoint activation, genome instability and cell death. In this study, we used C. elegans to determine the contribution of TLS activity on long-term stability of an animal genome. We monitored and compared the types of mutations that accumulate in REV1, REV3, POLH1 and POLK deficient animals that were grown under unchallenged conditions. We also addressed redundancies in TLS activity by combining all deficiencies. Remarkably, animals that are deficient for all Y-family polymerases as well as animals that have lost all TLS activity are viable and produce progeny, demonstrating that TLS is not essential for animal life. Whole genome sequencing analyses, however, reveal that TLS is needed to prevent genomic scars from accumulating. These scars, which are the product of polymerase theta-mediated end joining (TMEJ), are found overrepresented at guanine bases, consistent with TLS suppressing DNA double-strand breaks (DSBs) from occurring at replication-blocking guanine adducts. We found that in C. elegans, TLS across spontaneous damage is predominantly error free and anti-clastogenic, and thus ensures preservation of genetic information.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Reparo do DNA por Junção de Extremidades , DNA Polimerase Dirigida por DNA/genética , Instabilidade Genômica , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Mutação , Reprodução
17.
Nat Commun ; 11(1): 1591, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221299

RESUMO

Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex from Pyrococcus abyssi at 3.77 Å. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography, protein-protein interaction measurements, and activity assays - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Polα replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation and a processive phase during replication.


Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Archaea , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/metabolismo , Proteínas de Ligação a DNA/química , DNA Polimerase Dirigida por DNA/genética , Eucariotos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Pyrococcus abyssi/genética , Pyrococcus abyssi/metabolismo , Proteínas Recombinantes de Fusão
18.
Arq Neuropsiquiatr ; 78(3): 163-168, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32215459

RESUMO

Herpes simplex virus (HSV) is a cause of a severe disease of the central nervous system (CNS) in humans. The demonstration of specific antibodies in the cerebrospinal fluid (CSF) may contribute to the retrospective neurological diagnosis. However, the commercial immunological tests for HSV infection are for use in serum samples. OBJECTIVE: The aim of the present study was to adapt a commercial kit anti-HSV IgG used for serum samples to be performed with a CSF sample. METHODS: Forty CSF specimens from 38 patients with suspected CNS HSV infection were serially diluted for detecting anti-HSV IgG by enzyme immunoassay (EIA). The same samples were also analyzed with the polymerase chain reaction (PCR). RESULTS: The sensitivity of EIA test for HSV was 5% (dilution 1:40) and 65% (dilution 1:2) in CSF, and HSV DNA PCR was 15%. The combined analysis of EIA (dilution 1:2) and PCR increased the sensitivity up to 72.5%. The inflammatory CSF was associated with positive HSV PCR. CONCLUSIONS: We demonstrated the importance to adapt serological anti-HSV IgG EIA test for CSF assays to increase the accuracy of the analysis, considering the low concentration of specific antibodies in CSF.


Assuntos
Anticorpos Antivirais/líquido cefalorraquidiano , Líquido Cefalorraquidiano/virologia , Herpes Simples/diagnóstico , Herpes Simples/virologia , Simplexvirus/isolamento & purificação , Adulto , DNA Viral/genética , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases , Feminino , Herpes Simples/líquido cefalorraquidiano , Humanos , Masculino , Pessoa de Meia-Idade , Sistema Nervoso , Reação em Cadeia da Polimerase/métodos , Estudos Retrospectivos , Simplexvirus/genética , Proteínas Virais
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA