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1.
Methods Mol Biol ; 2444: 171-182, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35290638

RESUMO

Endonucleolytic cleavage of DNA ends by the human Mre11-Rad50-Nbs1 (MRN) complex occurs in a manner that is promoted by DNA-dependent protein kinase (DNA-PK). A method is described to isolate DNA-PK-bound fragments released from chromatin in human cells using a modified Gentle Lysis and Size Selection chromatin immunoprecipitation (GLASS-ChIP) protocol. This method, combined with real-time PCR or next-generation sequencing, can identify sites of MRN endonucleolytic cutting adjacent to DNA-PK binding sites in human cells.


Assuntos
Proteínas de Ligação a DNA , Proteínas Quinases , Imunoprecipitação da Cromatina , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Quinases/genética
2.
Methods Enzymol ; 661: 205-217, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34776213

RESUMO

Endonucleolytic cleavage of DNA ends by the human Mre11-Rad50-Nbs1 (MRN) complex occurs in a manner that is promoted by DNA-dependent Protein Kinase (DNA-PK). A method is described to isolate DNA-PK-bound fragments released from chromatin in human cells using a modified Gentle Lysis and Size Selection chromatin immunoprecipitation (GLASS-ChIP) protocol. This method, combined with real-time PCR or next-generation sequencing, can identify sites of MRN endonucleolytic cutting adjacent to DNA-PK binding sites in human cells.


Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Imunoprecipitação da Cromatina , DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteína Homóloga a MRE11/metabolismo , Proteínas Quinases/genética
3.
Nat Rev Mol Cell Biol ; 22(12): 796-814, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34429537

RESUMO

The protein kinase ataxia telangiectasia mutated (ATM) is a master regulator of double-strand DNA break (DSB) signalling and stress responses. For three decades, ATM has been investigated extensively to elucidate its roles in the DNA damage response (DDR) and in the pathogenesis of ataxia telangiectasia (A-T), a human neurodegenerative disease caused by loss of ATM. Although hundreds of proteins have been identified as ATM phosphorylation targets and many important roles for this kinase have been identified, it is still unclear how ATM deficiency leads to the early-onset cerebellar degeneration that is common in all individuals with A-T. Recent studies suggest the existence of links between ATM deficiency and other cerebellum-specific neurological disorders, as well as the existence of broader similarities with more common neurodegenerative disorders. In this Review, we discuss recent structural insights into ATM regulation, and possible aetiologies of A-T phenotypes, including reactive oxygen species, mitochondrial dysfunction, alterations in transcription, R-loop metabolism and alternative splicing, defects in cellular proteostasis and metabolism, and potential pathogenic roles for hyper-poly(ADP-ribosyl)ation.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ataxia Telangiectasia/metabolismo , Doenças Neurodegenerativas/metabolismo , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/patologia , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Reparo do DNA , Homeostase , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Oxirredução , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , RNA/metabolismo
4.
DNA Repair (Amst) ; 105: 103155, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34116476

RESUMO

The accumulation of unrepaired DNA lesions is associated with many pathological outcomes in humans, particularly in neurodegenerative diseases and in normal aging. Evidence supporting a causal role for DNA damage in the onset and progression of neurodegenerative disease has come from rare human patients with mutations in DNA damage response genes as well as from model organisms; however, the generality of this relationship in the normal population is unclear. In addition, the relevance of DNA damage in the context of proteotoxic stress-the widely accepted paradigm for pathology during neurodegeneration-is not well understood. Here, observations supporting intertwined roles of DNA damage and proteotoxicity in aging-related neurological outcomes are reviewed, with particular emphasis on recent insights into the relationships between DNA repair and autophagy, the ubiquitin proteasome system, formation of protein aggregates, poly-ADP-ribose polymerization, and transcription-driven DNA lesions.


Assuntos
Dano ao DNA , Reparo do DNA , Proteostase , Animais , Autofagia , DNA/metabolismo , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Poli Adenosina Difosfato Ribose/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo
5.
Dev Cell ; 56(4): 461-477.e7, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33621493

RESUMO

Homology-directed repair (HDR) safeguards DNA integrity under various forms of stress, but how HDR protects replicating genomes under extensive metabolic alterations remains unclear. Here, we report that besides stalling replication forks, inhibition of ribonucleotide reductase (RNR) triggers metabolic imbalance manifested by the accumulation of increased reactive oxygen species (ROS) in cell nuclei. This leads to a redox-sensitive activation of the ATM kinase followed by phosphorylation of the MRE11 nuclease, which in HDR-deficient settings degrades stalled replication forks. Intriguingly, nascent DNA degradation by the ROS-ATM-MRE11 cascade is also triggered by hypoxia, which elevates signaling-competent ROS and attenuates functional HDR without arresting replication forks. Under these conditions, MRE11 degrades daughter-strand DNA gaps, which accumulate behind active replisomes and attract error-prone DNA polymerases to escalate mutation rates. Thus, HDR safeguards replicating genomes against metabolic assaults by restraining mutagenic repair at aberrantly processed nascent DNA. These findings have implications for cancer evolution and tumor therapy.


Assuntos
Replicação do DNA , Genoma Humano , Metabolismo , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA2/deficiência , Proteína BRCA2/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , DNA/metabolismo , Humanos , Proteína Homóloga a MRE11/metabolismo , Modelos Biológicos , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Polimerização , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
6.
Mol Cell ; 81(7): 1515-1533.e5, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33571423

RESUMO

Loss of the ataxia-telangiectasia mutated (ATM) kinase causes cerebellum-specific neurodegeneration in humans. We previously demonstrated that deficiency in ATM activation via oxidative stress generates insoluble protein aggregates in human cells, reminiscent of protein dysfunction in common neurodegenerative disorders. Here, we show that this process is driven by poly-ADP-ribose polymerases (PARPs) and that the insoluble protein species arise from intrinsically disordered proteins associating with PAR-associated genomic sites in ATM-deficient cells. The lesions implicated in this process are single-strand DNA breaks dependent on reactive oxygen species, transcription, and R-loops. Human cells expressing Mre11 A-T-like disorder mutants also show PARP-dependent aggregation identical to ATM deficiency. Lastly, analysis of A-T patient cerebellum samples shows widespread protein aggregation as well as loss of proteins known to be critical in human spinocerebellar ataxias that is not observed in neocortex tissues. These results provide a hypothesis accounting for loss of protein integrity and cerebellum function in A-T.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Quebras de DNA de Cadeia Simples , Proteína Homóloga a MRE11/deficiência , Neocórtex/metabolismo , Poli ADP Ribosilação , Proteostase , Ataxias Espinocerebelares/metabolismo , Adulto , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Neocórtex/patologia , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia
7.
Methods Mol Biol ; 2153: 59-69, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840772

RESUMO

DNA double-strand break (DSB) end resection initiates homologous recombination (HR) and is critical for genomic stability. DSB resection has been monitored indirectly in mammalian cells using detection of protein foci or BrdU foci formation, which is dependent on single-stranded DNA (ssDNA) products of resection. Here we describe a quantitative PCR (qPCR)-based assay to directly measure levels of ssDNA intermediates generated by resection at specific DSB sites in human cells, which is more quantitative and precise with respect to the extent and efficiency of resection compared with previous methods. This assay, excluding the time for making the stable cell line expressing the restriction enzyme AsiSI fused to the estrogen receptor hormone-binding domain (ER-AsiSI), can be completed within 3 days.


Assuntos
Quebras de DNA de Cadeia Dupla , Enzimas de Restrição do DNA/genética , Receptores de Estrogênio/genética , Reparo de DNA por Recombinação , Sítios de Ligação , Técnicas de Cultura de Células , Enzimas de Restrição do DNA/metabolismo , Instabilidade Genômica , Células HEK293 , Humanos , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo
8.
Cancer Res ; 81(2): 426-437, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33239428

RESUMO

ATM kinase is a tumor suppressor and a master regulator of the DNA damage response. Most cancer-associated alterations to ATM are missense mutations at the PI3-kinase regulatory domain (PRD) or the kinase domain. Expression of kinase-dead (KD) ATM protein solely accelerates lymphomagenesis beyond ATM loss. To understand how PRD suppresses lymphomagenesis, we introduced the cancer-associated PRD mutation R3008H (R3016 in mouse) into mice. R3008H abrogated DNA damage- and oxidative stress-induced activation of ATM without consistently affecting ATM protein stability and recruitment. In contrast to the early embryonic lethality of AtmKD/KD mice, AtmR3016H (AtmR/R ) mice were viable, immunodeficient, and displayed spontaneous craniofacial abnormalities and delayed lymphomagenesis compared with Atm-/- controls. Mechanistically, R3008H rescued the tardy exchange of ATM-KD at DNA damage foci, indicating that PRD coordinates ATM activation with its exchange at DNA-breaks. Taken together, our results reveal a unique tumorigenesis profile for PRD mutations that is distinct from null or KD mutations. SIGNIFICANT: This study functionally characterizes the most common ATM missense mutation R3008H in cancer and identifies a unique role of PI3-kinase regulatory domain in ATM activation.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Dano ao DNA , Mutação , Neoplasias/genética , Animais , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Estimativa de Kaplan-Meier , Linfócitos/metabolismo , Linfócitos/patologia , Camundongos Knockout , Camundongos Transgênicos , Neoplasias/metabolismo
9.
EMBO Rep ; 22(1): e50500, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33245190

RESUMO

The denitrosylase S-nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S-nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhibitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox-mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox-insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T-cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function.


Assuntos
Aldeído Oxirredutases , Mitofagia , Aldeído Oxirredutases/metabolismo , Senescência Celular , Oxirredução , Estresse Oxidativo/genética
10.
Curr Opin Genet Dev ; 71: 55-62, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34293662

RESUMO

DNA double-strand breaks can be repaired through ligation-based pathways (non-homologous end-joining) or replication-based pathways (homologous recombination) in eukaryotic cells. The decisions that govern these outcomes are widely viewed as a competition between factors that recognize DNA ends and physically promote association of factors specific to each pathway, commonly known as 'pathway choice'. Here I review recent results in the literature and propose that this decision is better described as a sequential set of binding and end processing events, with non-homologous end joining as the first decision point. Physical association and co-localization of end resection factors with non-homologous end-joining factors suggests that ends are transferred between these complexes, thus the ultimate outcome is not the result of a competition but is more akin to a relay race that is determined by the efficiency of the initial end-joining event and the availability of activated DNA end-processing enzymes.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Animais , DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Recombinação Homóloga , Mamíferos/genética
11.
PLoS Biol ; 18(7): e3000606, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32687490

RESUMO

The 70 kDa heat shock protein (HSP70) family of chaperones are the front line of protection from stress-induced misfolding and aggregation of polypeptides in most organisms and are responsible for promoting the stability, folding, and degradation of clients to maintain cellular protein homeostasis. Here, we demonstrate quantitative identification of HSP70 and 71 kDa heat shock cognate (HSC70) clients using a ubiquitin-mediated proximity tagging strategy and show that, despite their high degree of similarity, these enzymes have largely nonoverlapping specificities. Both proteins show a preference for association with newly synthesized polypeptides, but each responds differently to changes in the stoichiometry of proteins in obligate multi-subunit complexes. In addition, expression of an amyotrophic lateral sclerosis (ALS)-associated superoxide dismutase 1 (SOD1) mutant protein induces changes in HSP70 and HSC70 client association and aggregation toward polypeptides with predicted disorder, indicating that there are global effects from a single misfolded protein that extend to many clients within chaperone networks. Together these findings show that the ubiquitin-activated interaction trap (UBAIT) fusion system can efficiently isolate the complex interactome of HSP chaperone family proteins under normal and stress conditions.


Assuntos
Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteoma/metabolismo , Linhagem Celular , Humanos , Mutação/genética , Ligação Proteica , Biossíntese de Proteínas , Dobramento de Proteína , Especificidade por Substrato , Ubiquitina/metabolismo
12.
Redox Biol ; 32: 101511, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32244177

RESUMO

The Ataxia-telangiectasia mutated (ATM) kinase responds to DNA double-strand breaks and other forms of cellular stress, including reactive oxygen species (ROS). Recent work in the field has uncovered links between mitochondrial ROS and ATM activation, suggesting that ATM acts as a sensor for mitochondrial derived ROS and regulates ROS accumulation in cells through this pathway. In addition, characterization of cells from Ataxia-telangiectasia patients as well as ATM-deficient mice and cell models suggest a role for ATM in modulating mitochondrial gene expression and function. Here we review ROS responses related to ATM function, recent evidence for ATM roles in mitochondrial maintenance and turnover, and the relationship between ATM and regulation of protein homeostasis.


Assuntos
Ataxia Telangiectasia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
13.
Mol Cell Biol ; 40(12)2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32205407

RESUMO

Maintenance of protein homeostasis in eukaryotes under normal growth and stress conditions requires the functions of Hsp70 chaperones and associated cochaperones. Here, we investigate an evolutionarily conserved serine phosphorylation that occurs at the site of communication between the nucleotide-binding and substrate-binding domains of Hsp70. Ser151 phosphorylation in yeast Hsp70 (Ssa1) is promoted by cyclin-dependent kinase (Cdk1) during normal growth. Phosphomimetic substitutions at this site (S151D) dramatically downregulate heat shock responses, a result conserved with HSC70 S153 in human cells. Phosphomimetic forms of Ssa1 also fail to relocalize in response to starvation conditions, do not associate in vivo with Hsp40 cochaperones Ydj1 and Sis1, and do not catalyze refolding of denatured proteins in vitro in cooperation with Ydj1 and Hsp104. Despite these negative effects on HSC70/HSP70 function, the S151D phosphomimetic allele promotes survival of heavy metal exposure and suppresses the Sup35-dependent [PSI+ ] prion phenotype, consistent with proposed roles for Ssa1 and Hsp104 in generating self-nucleating seeds of misfolded proteins. Taken together, these results suggest that Cdk1 can downregulate Hsp70 function through phosphorylation of this site, with potential costs to overall chaperone efficiency but also advantages with respect to reduction of metal-induced and prion-dependent protein aggregate production.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Sítios de Ligação , Linhagem Celular , Proteínas de Choque Térmico HSC70/química , Proteínas de Choque Térmico HSP70/química , Humanos , Metais Pesados/metabolismo , Fosforilação , Agregados Proteicos , Desnaturação Proteica , Domínios Proteicos , Dobramento de Proteína , Proteostase , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Estresse Fisiológico
14.
Nat Commun ; 11(1): 857, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051414

RESUMO

Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm-/- spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.


Assuntos
Endodesoxirribonucleases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Recombinação Homóloga/fisiologia , Meiose/fisiologia , Espermatócitos/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Feminino , Histona-Lisina N-Metiltransferase/genética , Proteína Homóloga a MRE11/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
15.
Sci Adv ; 6(2): eaay0922, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31934630

RESUMO

The repair of DNA double-strand breaks occurs through nonhomologous end joining or homologous recombination in vertebrate cells-a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here, we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK-bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from nonhomologous end joining to homologous recombination by facilitating DNA-PK removal.


Assuntos
Proteína Quinase Ativada por DNA/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Complexos Multiproteicos/metabolismo , Linhagem Celular , Humanos , Imagem Individual de Molécula
16.
Genes Dev ; 33(23-24): 1751-1774, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31753913

RESUMO

Bromodomain proteins (BRD) are key chromatin regulators of genome function and stability as well as therapeutic targets in cancer. Here, we systematically delineate the contribution of human BRD proteins for genome stability and DNA double-strand break (DSB) repair using several cell-based assays and proteomic interaction network analysis. Applying these approaches, we identify 24 of the 42 BRD proteins as promoters of DNA repair and/or genome integrity. We identified a BRD-reader function of PCAF that bound TIP60-mediated histone acetylations at DSBs to recruit a DUB complex to deubiquitylate histone H2BK120, to allowing direct acetylation by PCAF, and repair of DSBs by homologous recombination. We also discovered the bromo-and-extra-terminal (BET) BRD proteins, BRD2 and BRD4, as negative regulators of transcription-associated RNA-DNA hybrids (R-loops) as inhibition of BRD2 or BRD4 increased R-loop formation, which generated DSBs. These breaks were reliant on topoisomerase II, and BRD2 directly bound and activated topoisomerase I, a known restrainer of R-loops. Thus, comprehensive interactome and functional profiling of BRD proteins revealed new homologous recombination and genome stability pathways, providing a framework to understand genome maintenance by BRD proteins and the effects of their pharmacological inhibition.


Assuntos
Instabilidade Genômica , Estruturas R-Loop , Reparo de DNA por Recombinação/genética , Fatores de Transcrição/genética , Acetilação , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Células HEK293 , Células HeLa , Humanos , Transativadores/metabolismo , Fatores de Transcrição/análise , Ubiquitinação , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
17.
Crit Rev Biochem Mol Biol ; 54(4): 371-384, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31577154

RESUMO

The repair of DNA double-strand breaks occurs through a series of defined steps that are evolutionarily conserved and well-understood in most experimental organisms. However, it is becoming increasingly clear that repair does not occur in isolation from other DNA transactions. Transcription of DNA produces topological changes, RNA species, and RNA-dependent protein complexes that can dramatically influence the efficiency and outcomes of DNA double-strand break repair. The transcription-associated history of several double-strand break repair factors is reviewed here, with an emphasis on their roles in regulating R-loops and the emerging role of R-loops in coordination of repair events. Evidence for nucleolytic processing of R-loops is also discussed, as well as the molecular tools commonly used to measure RNA-DNA hybrids in cells.


Assuntos
Reparo do DNA/genética , DNA/genética , Estruturas R-Loop/genética , RNA/genética , Transcrição Genética , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA , DNA Helicases/genética , Endodesoxirribonucleases/genética , Humanos , RNA Helicases/genética , Recombinação Genética
18.
Mol Cell ; 75(1): 145-153.e5, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31153714

RESUMO

Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded DNA (ssDNA) overhangs. Resection regulation in bacteria is programmed by a DNA sequence, but a general mechanism limiting resection in eukaryotes has remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify phosphorylated RPA (pRPA) as a negative resection regulator. Bloom's syndrome (BLM) helicase together with exonuclease 1 (EXO1) and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which further stimulates DNA resection. RPA is phosphorylated during resection as part of the DNA damage response (DDR). Remarkably, pRPA inhibits DNA resection in cellular assays and in vitro via inhibition of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA provides a feedback loop between DNA resection and the DDR.


Assuntos
DNA de Cadeia Simples/genética , Retroalimentação Fisiológica , RecQ Helicases/genética , Proteínas Recombinantes de Fusão/genética , Proteína de Replicação A/genética , Sítios de Ligação , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica , Recombinação Homóloga , Humanos , Microscopia de Fluorescência , Nucleossomos/química , Nucleossomos/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fosforilação , Ligação Proteica , RecQ Helicases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula
19.
Methods Mol Biol ; 2004: 269-287, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31147924

RESUMO

The Mre11-Rad50-Nbs1 (MRN) complex coordinates the repair of DNA double-strand breaks, replication fork restart, meiosis, class-switch recombination, and telomere maintenance. As such, MRN is an essential molecular machine that has homologs in all organisms of life, from bacteriophage to humans. In human cells, MRN is a >500 kDa multifunctional complex that encodes DNA binding, ATPase, and both endonuclease and exonuclease activities. MRN also forms larger assemblies and interacts with multiple DNA repair and replication factors. The enzymatic properties of MRN have been the subject of intense research for over 20 years, and more recently, single-molecule biophysics studies are beginning to probe its many biochemical activities. Here, we describe the methods used to overexpress, fluorescently label, and visualize MRN and its activities on single molecules of DNA.


Assuntos
Hidrolases Anidrido Ácido/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/metabolismo , Adenosina Trifosfatases/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Humanos , Meiose/fisiologia
20.
Elife ; 72018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30523780

RESUMO

The Sae2/CtIP protein is required for efficient processing of DNA double-strand breaks that initiate homologous recombination in eukaryotic cells. Sae2/CtIP is also important for survival of single-stranded Top1-induced lesions and CtIP is known to associate directly with transcription-associated complexes in mammalian cells. Here we investigate the role of Sae2/CtIP at single-strand lesions in budding yeast and in human cells and find that depletion of Sae2/CtIP promotes the accumulation of stalled RNA polymerase and RNA-DNA hybrids at sites of highly expressed genes. Overexpression of the RNA-DNA helicase Senataxin suppresses DNA damage sensitivity and R-loop accumulation in Sae2/CtIP-deficient cells, and a catalytic mutant of CtIP fails to complement this sensitivity, indicating a role for CtIP nuclease activity in the repair process. Based on this evidence, we propose that R-loop processing by 5' flap endonucleases is a necessary step in the stabilization and removal of nascent R-loop initiating structures in eukaryotic cells.


Assuntos
Endonucleases/genética , Células Eucarióticas/metabolismo , Recombinação Homóloga/genética , RNA Helicases/genética , Proteínas de Saccharomyces cerevisiae/genética , Catálise , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , DNA Helicases , Reparo do DNA/genética , DNA Topoisomerases Tipo I/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Enzimas Multifuncionais , Saccharomyces cerevisiae/genética
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