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1.
Cell ; 186(22): 4898-4919.e25, 2023 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-37827155

RESUMO

Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.


Assuntos
Proteína de Replicação A , Expansão das Repetições de Trinucleotídeos , Animais , Humanos , Camundongos , DNA/genética , Reparo de Erro de Pareamento de DNA , Doença de Huntington/genética , Proteínas/genética , Ataxias Espinocerebelares/genética , Proteína de Replicação A/metabolismo
2.
Mol Cell ; 65(2): 272-284, 2017 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-28107649

RESUMO

The histone chaperone HIRA is involved in depositing histone variant H3.3 into distinct genic regions, including promoters, enhancers, and gene bodies. However, how HIRA deposits H3.3 to these regions remains elusive. Through a short hairpin RNA (shRNA) screening, we identified single-stranded DNA binding protein replication protein A (RPA) as a regulator of the deposition of newly synthesized H3.3 into chromatin. We show that RPA physically interacts with HIRA to form RPA-HIRA-H3.3 complexes, and it co-localizes with HIRA and H3.3 at gene promoters and enhancers. Depletion of RPA1, the largest subunit of the RPA complex, dramatically reduces both HIRA association with chromatin and the deposition of newly synthesized H3.3 at promoters and enhancers and leads to altered transcription at gene promoters. These results support a model whereby RPA, best known for its role in DNA replication and repair, recruits HIRA to promoters and enhancers and regulates deposition of newly synthesized H3.3 to these regulatory elements for gene regulation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Elementos Facilitadores Genéticos , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Regiões Promotoras Genéticas , Proteína de Replicação A/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Cromatina/genética , DNA/genética , Proteínas de Ligação a DNA/genética , Fase G1 , Células HEK293 , Células HeLa , Chaperonas de Histonas/genética , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Proteína de Replicação A/genética , Fatores de Transcrição/genética , Transfecção
3.
Blood ; 139(7): 1039-1051, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-34767620

RESUMO

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.


Assuntos
Transtornos da Insuficiência da Medula Óssea/patologia , Mutação com Ganho de Função , Heterozigoto , Síndromes Mielodisplásicas/patologia , Proteína de Replicação A/genética , Encurtamento do Telômero , Telômero/genética , Adolescente , Adulto , Transtornos da Insuficiência da Medula Óssea/etiologia , Transtornos da Insuficiência da Medula Óssea/metabolismo , Diferenciação Celular , Criança , Feminino , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/etiologia , Síndromes Mielodisplásicas/metabolismo , Adulto Jovem
4.
Nucleic Acids Res ; 46(12): 6238-6256, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29788478

RESUMO

Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj-/- cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj-/- cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Afidicolina/toxicidade , Linhagem Celular , Quinase 1 do Ponto de Checagem/metabolismo , Galinhas , Cisplatino/toxicidade , DNA de Cadeia Simples , Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Quadruplex G , Mutação de Sentido Incorreto , Oxazóis/toxicidade , RNA Helicases/química , Rad51 Recombinase/análise , Recombinases/genética , Recombinases/metabolismo , Proteína de Replicação A/metabolismo , Estresse Fisiológico
5.
Proc Natl Acad Sci U S A ; 113(9): E1170-9, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26884156

RESUMO

Exonuclease 1 (Exo1) is a 5'→3' exonuclease and 5'-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1-a recently identified human SSB that promotes DNA resection during homologous recombination-supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.


Assuntos
Enzimas Reparadoras do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Exodesoxirribonucleases/fisiologia , Biocatálise , Dano ao DNA , Humanos , Saccharomyces cerevisiae/metabolismo
6.
Biochim Biophys Acta Gen Subj ; 1862(6): 1482-1491, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29550431

RESUMO

BACKGROUND: Expansion of the C9orf72 hexanucleotide repeat (GGGGCC)n·(GGCCCC)n is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both strands of the C9orf72 repeat have been shown to form unusual DNA and RNA structures that are thought to be involved in mutagenesis and/or pathogenesis. We previously showed that the C-rich DNA strands from the C9orf72 repeat can form four-stranded quadruplexes at neutral pH. The cytosine residues become protonated under slightly acidic pH (pH 4.5-6.2), facilitating the formation of intercalated i-motif structures. METHODS: Using CD spectroscopy, UV melting, and gel electrophoresis, we demonstrate a pH-induced structural transition of the C-rich DNA strand of the C9orf72 repeat at pHs reported to exist in living cells under stress, including during neurodegeneration and cancer. RESULTS: We show that the repeats with lengths of 4, 6, and 8 units, form intercalated quadruplex i-motifs at low pH (pH < 5) and monomolecular hairpins and monomolecular quadruplexes under neutral-basic conditions (pH ≥ 8). Furthermore, we show that the human replication protein A (RPA) binds to the G-rich and C-rich DNA strands under acidic conditions, suggesting that it can bind to i-motif structures. CONCLUSIONS: In the proper sequence context, i-motif structures can form at pH values found in some cells in vivo. GENERAL SIGNIFICANCE: DNA conformational plasticity exists over broad range of solution conditions.


Assuntos
Ácidos/química , Proteína C9orf72/química , Citosina/química , Expansão das Repetições de DNA , Quadruplex G , Estresse Fisiológico , Humanos , Concentração de Íons de Hidrogênio , Conformação de Ácido Nucleico
7.
Nucleic Acids Res ; 44(12): 5758-72, 2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27131385

RESUMO

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA-DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions.


Assuntos
Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/genética , Rad51 Recombinase/genética , Proteínas Recombinantes de Fusão/genética , Proteína de Replicação A/genética , Sítios de Ligação , Clonagem Molecular , Quebras de DNA de Cadeia Simples , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Cinética , Simulação de Dinâmica Molecular , Mutação , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Rad51 Recombinase/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Replicação A/química , Proteína de Replicação A/metabolismo , Imagem Individual de Molécula , Gravação em Vídeo
8.
Bioessays ; 36(12): 1156-61, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25171654

RESUMO

Replication protein A (RPA), the major single-stranded DNA-binding protein in eukaryotic cells, is required for processing of single-stranded DNA (ssDNA) intermediates found in replication, repair, and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high-affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies showed that while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error-prone repair of double-stranded breaks and promote error-free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis.


Assuntos
Reparo do DNA , Replicação do DNA , DNA Fúngico/metabolismo , DNA de Cadeia Simples/metabolismo , Regulação Fúngica da Expressão Gênica , Proteína de Replicação A/química , Ustilago/metabolismo , Ligação Competitiva , DNA Fúngico/genética , DNA de Cadeia Simples/genética , Modelos Moleculares , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Recombinação Genética , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Transdução de Sinais , Ustilago/genética
9.
J Biol Chem ; 289(29): 19928-41, 2014 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-24895130

RESUMO

Understanding how cellular machinery deals with chromosomal genome complexity is an important question because protein bound to DNA may affect various cellular processes of nucleic acid metabolism. DNA helicases are at the forefront of such processes, yet there is only limited knowledge how they remodel protein-DNA complexes and how these mechanisms are regulated. We have determined that representative human RecQ and Fe-S cluster DNA helicases are potently blocked by a protein-DNA interaction. The Fanconi anemia group J (FANCJ) helicase partners with the single-stranded DNA-binding protein replication protein A (RPA) to displace BamHI-E111A bound to duplex DNA in a specific manner. Protein displacement was dependent on the ATPase-driven function of the helicase and unique properties of RPA. Further biochemical studies demonstrated that the shelterin proteins TRF1 and TRF2, which preferentially bind the telomeric repeat found at chromosome ends, effectively block FANCJ from unwinding the forked duplex telomeric substrate. RPA, but not the Escherichia coli single-stranded DNA-binding protein or shelterin factor Pot1, stimulated FANCJ ejection of TRF1 from the telomeric DNA substrate. FANCJ was also able to displace TRF2 from the telomeric substrate in an RPA-dependent manner. The stimulation of helicase-catalyzed protein displacement is also observed with the DNA helicase RECQ1, suggesting a conserved functional interaction of RPA-interacting helicases. These findings suggest that partnerships between RPA and interacting human DNA helicases may greatly enhance their ability to dislodge proteins bound to duplex DNA, an activity that is likely to be highly relevant to their biological roles in DNA metabolism.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , DNA/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , RecQ Helicases/metabolismo , Proteína de Replicação A/metabolismo , Substituição de Aminoácidos , Sequência de Bases , DNA/química , DNA/genética , Desoxirribonuclease BamHI/metabolismo , Exodesoxirribonucleases/metabolismo , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Replicação A/genética , Especificidade por Substrato , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Helicase da Síndrome de Werner
10.
J Biol Chem ; 287(6): 3908-18, 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22179778

RESUMO

Replication protein A (RPA), the major eukaryotic single-strand DNA (ssDNA)-binding protein, is essential for replication, repair, recombination, and checkpoint activation. Defects in RPA-associated cellular activities lead to genomic instability, a major factor in the pathogenesis of cancer and other diseases. ssDNA binding activity is primarily mediated by two domains in the 70-kDa subunit of the RPA complex. These ssDNA interactions are mediated by a combination of polar residues and four conserved aromatic residues. Mutation of the aromatic residues causes a modest decrease in binding to long (30-nucleotide) ssDNA fragments but results in checkpoint activation and cell cycle arrest in cells. We have used a combination of biochemical analysis and knockdown replacement studies in cells to determine the contribution of these aromatic residues to RPA function. Cells containing the aromatic residue mutants were able to progress normally through S-phase but were defective in DNA repair. Biochemical characterization revealed that mutation of the aromatic residues severely decreased binding to short ssDNA fragments less than 20 nucleotides long. These data indicate that altered binding of RPA to short ssDNA intermediates causes a defect in DNA repair but not in DNA replication. These studies show that cells require different RPA functions in DNA replication and DNA repair.


Assuntos
Reparo do DNA/fisiologia , DNA de Cadeia Simples/metabolismo , Proteína de Replicação A/metabolismo , Substituição de Aminoácidos , Animais , Pontos de Checagem do Ciclo Celular/fisiologia , Replicação do DNA/fisiologia , DNA de Cadeia Simples/genética , Drosophila melanogaster , Células HeLa , Humanos , Camundongos , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Proteína de Replicação A/genética , Fase S/fisiologia , Saccharomyces cerevisiae
11.
J Biol Chem ; 287(43): 36123-31, 2012 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-22948311

RESUMO

Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair.


Assuntos
Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/química , Proteínas Serina-Treonina Quinases/química , Proteína de Replicação A/química , Transdução de Sinais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Quinase 1 do Ponto de Checagem , Reparo do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos , Mutação , Fosforilação/fisiologia , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
12.
J Biol Chem ; 287(51): 42773-83, 2012 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-23095756

RESUMO

Previously, we characterized Saccharomyces cerevisiae exonuclease 5 (EXO5), which is required for mitochondrial genome maintenance. Here, we identify the human homolog (C1orf176; EXO5) that functions in the repair of nuclear DNA damage. Human EXO5 (hEXO5) contains an iron-sulfur cluster. It is a single-stranded DNA (ssDNA)-specific bidirectional exonuclease with a strong preference for 5'-ends. After loading at an ssDNA end, hEXO5 slides extensively along the ssDNA prior to cutting, hence the designation sliding exonuclease. However, the single-stranded binding protein human replication protein A (hRPA) restricts sliding and enforces a unique, species-specific 5'-directionality onto hEXO5. This specificity is lost with a mutant form of hRPA (hRPA-t11) that fails to interact with hEXO5. hEXO5 localizes to nuclear repair foci in response to DNA damage, and its depletion in human cells leads to an increased sensitivity to DNA-damaging agents, in particular interstrand cross-linking-inducing agents. Depletion of hEXO5 also results in an increase in spontaneous and damage-induced chromosome abnormalities including the frequency of triradial chromosomes, suggesting an additional defect in the resolution of stalled DNA replication forks in hEXO5-depleted cells.


Assuntos
Exonucleases/metabolismo , Genoma Humano/genética , Instabilidade Genômica , Sequência de Aminoácidos , Biocatálise/efeitos dos fármacos , Biocatálise/efeitos da radiação , Aberrações Cromossômicas/efeitos dos fármacos , Aberrações Cromossômicas/efeitos da radiação , Sequência Conservada , Reagentes de Ligações Cruzadas/farmacologia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , DNA de Cadeia Simples/metabolismo , Exonucleases/química , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/efeitos da radiação , Humanos , Proteínas Ferro-Enxofre/metabolismo , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/efeitos da radiação , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos da radiação , Proteína de Replicação A/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/efeitos da radiação , Raios Ultravioleta
13.
Proc Natl Acad Sci U S A ; 107(31): 13660-5, 2010 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-20616048

RESUMO

ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase.


Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Replicação A/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/isolamento & purificação , Quinase 1 do Ponto de Checagem , Enzimas Reparadoras do DNA/isolamento & purificação , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/isolamento & purificação
14.
J Biol Chem ; 286(5): 3497-508, 2011 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-21107010

RESUMO

The premature aging and cancer-prone disease Werner syndrome is caused by loss of function of the RecQ helicase family member Werner syndrome protein (WRN). At the cellular level, loss of WRN results in replication abnormalities and chromosomal aberrations, indicating that WRN plays a role in maintenance of genome stability. Consistent with this notion, WRN possesses annealing, exonuclease, and ATPase-dependent helicase activity on DNA substrates, with particularly high affinity for and activity on replication and recombination structures. After certain DNA-damaging treatments, WRN is recruited to sites of blocked replication and co-localizes with the human single-stranded DNA-binding protein replication protein A (RPA). In this study we examined the physical and functional interaction between WRN and RPA specifically in relation to replication fork blockage. Co-immunoprecipitation experiments demonstrated that damaging treatments that block DNA replication substantially increased association between WRN and RPA in vivo, and a direct interaction between purified WRN and RPA was confirmed. Furthermore, we examined the combined action of RPA (unmodified and hyperphosphorylation mimetic) and WRN on model replication fork and gapped duplex substrates designed to bind RPA. Even with RPA bound stoichiometrically to this gap, WRN efficiently catalyzed regression of the fork substrate. Further analysis showed that RPA could be displaced from both substrates by WRN. RPA displacement by WRN was independent of its ATPase- and helicase-dependent remodeling of the fork. Taken together, our results suggest that, upon replication blockage, WRN and RPA functionally interact and cooperate to help properly resolve replication forks and maintain genome stability.


Assuntos
Replicação do DNA , Exodesoxirribonucleases/fisiologia , RecQ Helicases/fisiologia , Proteína de Replicação A/fisiologia , Adenosina Trifosfatases , Dano ao DNA , DNA Helicases , Exodesoxirribonucleases/metabolismo , Instabilidade Genômica , Humanos , Ligação Proteica , RecQ Helicases/metabolismo , Proteína de Replicação A/metabolismo , Helicase da Síndrome de Werner
15.
Nucleic Acids Res ; 38(3): 846-58, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19942684

RESUMO

Replication Protein A (RPA) is a single-stranded DNA-binding protein essential for DNA replication, repair, recombination and cell-cycle regulation. A human homolog of the RPA2 subunit, called RPA4, was previously identified and shown to be expressed in colon mucosal and placental cells; however, the function of RPA4 was not determined. To examine the function of RPA4 in human cells, we carried out knockdown and replacement studies to determine whether RPA4 can substitute for RPA2 in the cell. Unlike RPA2, exogenous RPA4 expression did not support chromosomal DNA replication and lead to cell-cycle arrest in G2/M. In addition, RPA4 localized to sites of DNA repair and reduced gamma-H2AX caused by RPA2 depletion. These studies suggest that RPA4 cannot support cell proliferation but can support processes that maintain the genomic integrity of the cell.


Assuntos
Ciclo Celular , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Apoptose , Reparo do DNA , Proteínas de Ligação a DNA/química , Genômica , Células HeLa , Humanos , Dados de Sequência Molecular , Fenótipo , Subunidades Proteicas/metabolismo , Proteína de Replicação A/antagonistas & inibidores , Proteína de Replicação A/metabolismo , Homologia de Sequência de Aminoácidos
16.
Nat Commun ; 13(1): 3743, 2022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35768435

RESUMO

Perturbation in the replication-stress response (RSR) and DNA-damage response (DDR) causes genomic instability. Genomic instability occurs in Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency disorder, yet the mechanism remains largely uncharacterized. Replication protein A (RPA), a single-strand DNA (ssDNA) binding protein, has key roles in the RSR and DDR. Here we show that human WAS-protein (WASp) modulates RPA functions at perturbed replication forks (RFs). Following genotoxic insult, WASp accumulates at RFs, associates with RPA, and promotes RPA:ssDNA complexation. WASp deficiency in human lymphocytes destabilizes RPA:ssDNA-complexes, impairs accumulation of RPA, ATR, ETAA1, and TOPBP1 at genotoxin-perturbed RFs, decreases CHK1 activation, and provokes global RF dysfunction. las17 (yeast WAS-homolog)-deficient S. cerevisiae also show decreased ScRPA accumulation at perturbed RFs, impaired DNA recombination, and increased frequency of DNA double-strand break (DSB)-induced single-strand annealing (SSA). Consequently, WASp (or Las17)-deficient cells show increased frequency of DSBs upon genotoxic insult. Our study reveals an evolutionarily conserved, essential role of WASp in the DNA stress-resolution pathway, such that WASp deficiency provokes RPA dysfunction-coupled genomic instability.


Assuntos
Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA de Cadeia Simples , Proteína de Replicação A , Proteínas de Saccharomyces cerevisiae , Proteína da Síndrome de Wiskott-Aldrich , Animais , Antígenos de Superfície/metabolismo , Reparo do DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Humanos , Ligação Proteica , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
17.
J Biol Chem ; 285(7): 4788-97, 2010 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-19996105

RESUMO

Replication protein A (RPA) is a heterotrimeric protein complex required for a large number of DNA metabolic processes, including DNA replication and repair. An alternative form of RPA (aRPA) has been described in which the RPA2 subunit (the 32-kDa subunit of RPA and product of the RPA2 gene) of canonical RPA is replaced by a homologous subunit, RPA4. The normal function of aRPA is not known; however, previous studies have shown that it does not support DNA replication in vitro or S-phase progression in vivo. In this work, we show that the RPA4 gene is expressed in normal human tissues and that its expression is decreased in cancerous tissues. To determine whether aRPA plays a role in cellular physiology, we investigated its role in DNA repair. aRPA interacted with both Rad52 and Rad51 and stimulated Rad51 strand exchange. We also showed that, by using a reconstituted reaction, aRPA can support the dual incision/excision reaction of nucleotide excision repair. aRPA is less efficient in nucleotide excision repair than canonical RPA, showing reduced interactions with the repair factor XPA and no stimulation of XPF-ERCC1 endonuclease activity. In contrast, aRPA exhibits higher affinity for damaged DNA than canonical RPA, which may explain its ability to substitute for RPA in the excision step of nucleotide excision repair. Our findings provide the first direct evidence for the function of aRPA in human DNA metabolism and support a model for aRPA functioning in chromosome maintenance functions in nonproliferating cells.


Assuntos
Reparo do DNA/fisiologia , Proteína de Replicação A/metabolismo , Reparo do DNA/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Técnicas In Vitro , Reação em Cadeia da Polimerase , Ligação Proteica/genética , Ligação Proteica/fisiologia , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteína de Replicação A/genética
18.
Nucleic Acids Res ; 37(7): 2313-26, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19244309

RESUMO

Replication protein A (RPA) is a heterotrimeric (70, 32 and 14 kDa subunits), single-stranded DNA-binding protein required for cellular DNA metabolism. All subunits of RPA are essential for life, but the specific functions of the 32 and 14 kDa subunits remains unknown. The 32 kDa subunit (RPA2) has multiple domains, but only the central DNA-binding domain (called DBD D) is essential for life in Saccharomyces cerevisiae. To define the essential function(s) of RPA2 in S. cerevisiae, a series of site-directed mutant forms of DBD D were generated. These mutant constructs were then characterized in vitro and in vivo. The mutations had minimal effects on the overall structure and activity of the RPA complex. However, several mutants were shown to disrupt crosslinking of RPA2 to DNA and to dramatically lower the DNA-binding affinity of a RPA2-containing subcomplex. When introduced into S. cerevisiae, all DBD D mutants were viable and supported normal growth rates and DNA replication. These findings indicate that RPA2-DNA interactions are not essential for viability and growth in S. cerevisiae. We conclude that DNA-binding activity of RPA2 is dispensable in yeast and that the essential function of DBD D is intra- and/or inter-protein interactions.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteína de Replicação A/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Proteína de Replicação A/química , Proteína de Replicação A/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Moldes Genéticos , Raios Ultravioleta
19.
Biochemistry ; 49(28): 5919-28, 2010 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-20545304

RESUMO

Replication protein A (RPA) is a single-stranded DNA-binding complex that is essential for DNA replication, repair, and recombination in eukaryotic cells. In addition to this canonical complex, we have recently characterized an alternative replication protein A complex (aRPA) that is unique to primates. aRPA is composed of three subunits: RPA1 and RPA3, also present in canonical RPA, and a primate-specific subunit RPA4, homologous to canonical RPA2. aRPA has biochemical properties similar to those of the canonical RPA complex but does not support DNA replication. We describe studies that aimed to identify what properties of aRPA prevent it from functioning in DNA replication. We show aRPA has weakened interaction with DNA polymerase alpha (pol alpha) and that aRPA is not able to efficiently stimulate DNA synthesis by pol alpha on aRPA-coated DNA. Additionally, we show that aRPA is unable to support de novo priming by pol alpha. Because pol alpha activity is essential for both initiation and Okazaki strand synthesis, we conclude that the inability of aRPA to support pol alpha loading causes aRPA to be defective in DNA replication. We also show that aRPA stimulates synthesis by DNA polymerase alpha in the presence of PCNA and RFC. This indicates that aRPA can support extension of DNA strands by DNA polymerase partial differential. This finding along with the previous observation that aRPA supports early steps of nucleotide excision repair and recombination indicates that aRPA can support DNA repair synthesis that requires polymerase delta, PCNA, and RFC and support a role for aRPA in DNA repair.


Assuntos
DNA Polimerase I/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Proteína de Replicação A/metabolismo , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transcrição Gênica
20.
J Biol Chem ; 284(27): 18458-70, 2009 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-19419957

RESUMO

FANCJ mutations are genetically linked to the Fanconi anemia complementation group J and predispose individuals to breast cancer. Understanding the role of FANCJ in DNA metabolism and how FANCJ dysfunction leads to tumorigenesis requires mechanistic studies of FANCJ helicase and its protein partners. In this work, we have examined the ability of FANCJ to unwind DNA molecules with specific base damage that can be mutagenic or lethal. FANCJ was inhibited by a single thymine glycol, but not 8-oxoguanine, in either the translocating or nontranslocating strands of the helicase substrate. In contrast, the human RecQ helicases (BLM, RECQ1, and WRN) display strand-specific inhibition of unwinding by the thymine glycol damage, whereas other DNA helicases (DinG, DnaB, and UvrD) are not significantly inhibited by thymine glycol in either strand. In the presence of replication protein A (RPA), but not Escherichia coli single-stranded DNA-binding protein, FANCJ efficiently unwound the DNA substrate harboring the thymine glycol damage in the nontranslocating strand; however, inhibition of FANCJ helicase activity by the translocating strand thymine glycol was not relieved. Strand-specific stimulation of human RECQ1 helicase activity was also observed, and RPA bound with high affinity to single-stranded DNA containing a single thymine glycol. Based on the biochemical studies, we propose a model for the specific functional interaction between RPA and FANCJ on the thymine glycol substrates. These studies are relevant to the roles of RPA, FANCJ, and other DNA helicases in the metabolism of damaged DNA that can interfere with basic cellular processes of DNA metabolism.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Dano ao DNA/fisiologia , DNA/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Estresse Oxidativo/genética , Proteína de Replicação A/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Neoplasias da Mama/genética , Adutos de DNA/genética , Adutos de DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Ativação Enzimática/fisiologia , Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Feminino , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Proteína de Replicação A/genética , Especificidade por Substrato , Timina/análogos & derivados , Timina/metabolismo
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