RESUMO
BACKGROUND: Freedom from rejection in pediatric heart transplant recipients is highly variable across centers. This study aimed to assess the center variation in methods used to diagnose rejection in the first-year post-transplant and determine the impact of this variation on patient outcomes. METHODS: The PHTS registry was queried for all rejection episodes in the first-year post-transplant (2010-2019). The primary method for rejection diagnosis was determined for each event as surveillance biopsy, echo diagnosis, or clinical. The percentage of first-year rejection events diagnosed by surveillance biopsy was used to approximate the surveillance strategy across centers. Methods of rejection diagnosis were described and patient outcomes were assessed based on surveillance biopsy utilization among centers. RESULTS: A total of 3985 patients from 56 centers were included. Of this group, 873 (22%) developed rejection within the first-year post-transplant. Surveillance biopsy was the most common method of rejection diagnosis (71.7%), but practices were highly variable across centers. The majority (73.6%) of first rejection events occurred within 3-months of transplantation. Diagnosis modality in the first-year was not independently associated with freedom from rejection, freedom from rejection with hemodynamic compromise, or overall graft survival. CONCLUSIONS: Rejection in the first-year after pediatric heart transplant occurs in 22% of patients and most commonly in the first 3 months post-transplant. Significant variation exists across centers in the methods used to diagnose rejection in pediatric heart transplant recipients, however, these variable strategies are not independently associated with freedom from rejection, rejection with hemodynamic compromise, or overall graft survival.
Assuntos
Rejeição de Enxerto/diagnóstico , Transplante de Coração/efeitos adversos , Padrões de Prática Médica , Adolescente , Fatores Etários , Criança , Feminino , Rejeição de Enxerto/etiologia , Humanos , Masculino , Sistema de Registros , Estudos Retrospectivos , Fatores de Risco , Fatores de TempoRESUMO
Allosensitized children listed with a requirement for a negative prospective crossmatch have high mortality. Previously, we found that listing with the intent to accept the first suitable organ offer, regardless of the possibility of a positive crossmatch (TAKE strategy), results in a survival advantage from the time of listing compared to awaiting transplantation across a negative crossmatch (WAIT). The cost-effectiveness of these strategies is unknown. We used Markov modeling to compare cost-effectiveness between these waitlist strategies for allosensitized children listed urgently for heart transplantation. We used registry data to estimate costs and waitlist/posttransplant outcomes. We assumed patients remained in hospital after listing, no positive crossmatches for WAIT, and a base-case probability of a positive crossmatch of 47% for TAKE. Accepting the first suitable organ offer cost less ($405 904 vs. $534 035) and gained more quality-adjusted life years (3.71 vs. 2.79). In sensitivity analyses, including substitution of waitlist data from children with unacceptable antigens specified during listing, TAKE remained cost-saving or cost-effective. Our findings suggest acceptance of the first suitable organ offer for urgently listed allosensitized pediatric heart transplant candidates is cost-effective and transplantation should not be denied because of allosensitization status alone.
Assuntos
Redução de Custos , Transplante de Coração/economia , Transplante de Coração/métodos , Teste de Histocompatibilidade/economia , Listas de Espera , Criança , Pré-Escolar , Estudos de Coortes , Análise Custo-Benefício , Bases de Dados Factuais , Emergências , Feminino , Rejeição de Enxerto , Sobrevivência de Enxerto , Transplante de Coração/efeitos adversos , Teste de Histocompatibilidade/métodos , Custos Hospitalares , Humanos , Lactente , Masculino , Cadeias de Markov , Seleção de Pacientes , Pediatria , Prognóstico , Sistema de Registros , Medição de Risco , Sensibilidade e Especificidade , Fatores de Tempo , Resultado do TratamentoRESUMO
Homologous recombination has a dual role in eukaryotic organisms. Firstly, it is responsible for the creation of genetic variability during meiosis by directing the formation of reciprocal crossovers that result in random combinations of alleles and traits. Secondly, in mitotic cells, it maintains the integrity of the genome by promoting the faithful repair of DNA double-strand breaks (DSBs). In vertebrates, it therefore plays a key role in tumour avoidance. Mutations in the tumour suppressor protein BRCA2 are associated with predisposition to breast and ovarian cancers, and loss of BRCA2 function leads to genetic instability. BRCA2 protein interacts directly with the RAD51 recombinase and regulates recombination-mediated DSB repair, accounting for the high levels of spontaneous chromosomal aberrations seen in BRCA2-defective cells. Recent observations indicate that BRCA2 also plays a critical role in meiotic recombination, this time through direct interactions with the meiosis-specific recombinase DMC1. The interactions of BRCA2 with RAD51 and DMC1 lead us to suggest that the BRCA2 tumour suppressor is a universal regulator of recombinase actions.
Assuntos
Proteína BRCA2/genética , Dano ao DNA/genética , Neoplasias/genética , Rad51 Recombinase/genética , Recombinação Genética/genética , Proteínas Reguladoras de Apoptose , Proteína BRCA2/biossíntese , Reparo do DNA/genética , Humanos , Meiose , Neoplasias/metabolismo , Rad51 Recombinase/metabolismoRESUMO
DNA nonhomologous end-joining (NHEJ) is the major pathway for repairing DNA double-strand breaks in mammalian cells. It also functions to carry out rearrangements at the specialized breaks introduced during V(D)J recombination. Here, we describe a patient with T(-)B(-) severe combined immunodeficiency, whose cells have defects closely resembling those of NHEJ-defective rodent cells. Cells derived from this patient show dramatic radiosensitivity, decreased double-strand break rejoining, and reduced fidelity in signal and coding joint formation during V(D)J recombination. Detailed examination indicates that the patient is defective neither in the known factors involved in NHEJ in mammals (Ku70, Ku80, DNA-dependent protein kinase catalytic subunit, Xrcc4, DNA ligase IV, or Artemis) nor in the Mre11/Rad50/Nbs1 complex, whose homologue in Saccharomyces cerevisiae functions in NHEJ. These results provide strong evidence that additional activities are crucial for NHEJ and V(D)J recombination in mammals.
Assuntos
DNA Nucleotidiltransferases/química , Reparo do DNA , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Animais , Domínio Catalítico , Células Cultivadas , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , DNA Nucleotidiltransferases/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta à Radiação , Fibroblastos/metabolismo , Humanos , Immunoblotting , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Células Tumorais Cultivadas , VDJ RecombinasesRESUMO
Cells defective in any of the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are sensitive to DNA cross-linking agents and to ionizing radiation. Because the paralogs are required for the assembly of DNA damage-induced RAD51 foci, and mutant cell lines are defective in homologous recombination and show genomic instability, their defect is thought to be caused by an inability to promote efficient recombinational repair. Here, we show that the five paralogs exist in two distinct complexes in human cells: one contains RAD51B, RAD51C, RAD51D, and XRCC2 (defined as BCDX2), whereas the other consists of RAD51C with XRCC3. Both protein complexes have been purified to homogeneity and their biochemical properties investigated. BCDX2 binds single-stranded DNA and single-stranded gaps in duplex DNA, in accord with the proposal that the paralogs play an early (pre-RAD51) role in recombinational repair. Moreover, BCDX2 complex binds specifically to nicks in duplex DNA. We suggest that the extreme sensitivity of paralog-defective cell lines to cross-linking agents is owing to defects in the processing of incised cross links and the consequential failure to initiate recombinational repair at these sites.
Assuntos
Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/isolamento & purificação , Testículo/química , Adenosina Trifosfatases/metabolismo , Baculoviridae/genética , Cromatografia em Gel , Reparo do DNA/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Microscopia Eletrônica , Testes de Precipitina , Ligação Proteica , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Rad51 Recombinase , Proteínas Recombinantes/metabolismo , Recombinação Genética , Testículo/citologiaRESUMO
Proteins that catalyse homologous recombination have been identified in all living organisms and are essential for the repair of damaged DNA as well as for the generation of genetic diversity. In bacteria homologous recombination is performed by the RecA protein, whereas in the eukarya a related protein called Rad51 is required to catalyse recombination and repair. More recently, archaeal homologues of RecA/Rad51 (RadA) have been identified and isolated. In this work we have cloned and purified the RadA protein from the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus and characterised its in vitro activities. We show that (i) RadA protein forms ring structures in solution and binds single- but not double-stranded DNA to form nucleoprotein filaments, (ii) RadA is a single-stranded DNA-dependent ATPase at elevated temperatures, and (iii) RadA catalyses efficient D-loop formation and strand exchange at temperatures of 60-70 degrees C. Finally, we have used electron microscopy to visualise RadA-mediated joint molecules, the intermediates of homologous recombination. Intriguingly, RadA shares properties of both the bacterial RecA and eukaryotic Rad51 recombinases.
Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nucleoproteínas/metabolismo , Recombinação Genética , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Archaeoglobus fulgidus/química , DNA/química , DNA/metabolismo , DNA/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Microscopia Eletrônica , Conformação de Ácido Nucleico , Nucleoproteínas/química , Nucleoproteínas/ultraestrutura , Ligação Proteica , Conformação Proteica , TemperaturaRESUMO
Double-strand breaks (DSBs) occur frequently during DNA replication. They are also caused by ionizing radiation, chemical damage or as part of the series of programmed events that occur during meiosis. In yeast, DSB repair requires RAD52, a protein that plays a critical role in homologous recombination. Here we describe the actions of human RAD52 protein in a model system for single-strand annealing (SSA) using tailed (i.e. exonuclease resected) duplex DNA molecules. Purified human RAD52 protein binds resected DSBs and promotes associations between complementary DNA termini. Heteroduplex intermediates of these recombination reactions have been visualized by electron microscopy, revealing the specific binding of multiple rings of RAD52 to the resected termini and the formation of large protein complexes at heteroduplex joints formed by RAD52-mediated annealing.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Recombinação Genética , Animais , Baculoviridae/metabolismo , Linhagem Celular , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Humanos , Insetos , Microscopia Eletrônica , Modelos Genéticos , Plasmídeos/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
Both the bacterial RecA protein and the eukaryotic Rad51 protein form helical nucleoprotein filaments on DNA that catalyze strand transfer between two homologous DNA molecules. However, only the ATP-binding cores of these proteins have been conserved, and this same core is also found within helicases and the F1-ATPase. The C-terminal domain of the RecA protein forms lobes within the helical RecA filament. However, the Rad51 proteins do not have the C-terminal domain found in RecA, but have an N-terminal extension that is absent in the RecA protein. Both the RecA C-terminal domain and the Rad51 N-terminal domain bind DNA. We have used electron microscopy to show that the lobes of the yeast and human Rad51 filaments appear to be formed by N-terminal domains. These lobes are conformationally flexible in both RecA and Rad51. Within RecA filaments, the change between the "active" and "inactive" states appears to mainly involve a large movement of the C-terminal lobe. The N-terminal domain of Rad51 and the C-terminal domain of RecA may have arisen from convergent evolution to play similar roles in the filaments.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Recombinases Rec A/química , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína , Rad51 RecombinaseRESUMO
In vertebrates, the RAD51 protein is required for genetic recombination, DNA repair, and cellular proliferation. Five paralogs of RAD51, known as RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3, have been identified and also shown to be required for recombination and genome stability. At the present time, however, very little is known about their biochemical properties or precise biological functions. As a first step toward understanding the roles of the RAD51 paralogs in recombination, the human RAD51C and XRCC3 proteins were overexpressed and purified from baculovirus-infected insect cells. The two proteins copurify as a complex, a property that reflects their endogenous association observed in HeLa cells. Purified RAD51C--XRCC3 complex binds single-stranded, but not duplex DNA, to form protein--DNA networks that have been visualized by electron microscopy.
Assuntos
Reparo do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/isolamento & purificação , Células HeLa , Humanos , Camundongos , Microscopia Eletrônica , Oligodesoxirribonucleotídeos/metabolismo , Coelhos , Rad51 Recombinase , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , SpodopteraRESUMO
Individuals carrying BRCA2 mutations are predisposed to breast and ovarian cancers. Here, we show that BRCA2 plays a dual role in regulating the actions of RAD51, a protein essential for homologous recombination and DNA repair. First, interactions between RAD51 and the BRC3 or BRC4 regions of BRCA2 block nucleoprotein filament formation by RAD51. Alterations to the BRC3 region that mimic cancer-associated BRCA2 mutations fail to exhibit this effect. Second, transport of RAD51 to the nucleus is defective in cells carrying a cancer-associated BRCA2 truncation. Thus, BRCA2 regulates both the intracellular localization and DNA binding ability of RAD51. Loss of these controls following BRCA2 inactivation may be a key event leading to genomic instability and tumorigenesis.
Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Recombinação Genética , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Proteína BRCA2 , Sítios de Ligação , Neoplasias da Mama/genética , Cromatografia em Gel , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Microscopia Eletrônica , Modelos Biológicos , Dados de Sequência Molecular , Peso Molecular , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Nucleoproteínas/antagonistas & inibidores , Nucleoproteínas/metabolismo , Nucleoproteínas/ultraestrutura , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Rad51 Recombinase , Frações Subcelulares , Especificidade por Substrato , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
A double-strand break in genomic DNA that remains unrepaired can be lethal for a cell. Indeed, the integrity of the genome is paramount for survival. It is therefore surprising that some cells deliberately introduce double-strand breaks at certain times during their life cycle. Why might they do this? What are the benefits? How are these breaks repaired? The answers to these questions lie in understanding the basis of meiotic recombination, the process that leads to genetic variation. This review summarizes the key roles played by the two recombinases, Dmc1 and Rad51, in the faithful repair of DNA breaks.
Assuntos
Proteínas de Ciclo Celular , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/química , Animais , DNA/ultraestrutura , Escherichia coli/enzimologia , Humanos , Meiose , Modelos Genéticos , Rad51 Recombinase , Recombinação GenéticaRESUMO
During homologous recombination, DNA strand exchange leads to Holliday junction formation. The movement, or branch migration, of this junction along DNA extends the length of the heteroduplex joint. In prokaryotes, branch migration and Holliday junction resolution are catalyzed by the RuvA and RuvB proteins, which form a complex with RuvC resolvase to form a "resolvasome". Mammalian cell-free extracts have now been fractionated to reveal analogous activities. An ATP-dependent branch migration activity, which migrates junctions through >2700 bp, cofractionates with the Holliday junction resolvase during several chromatographic steps. Together, the two activities promote concerted branch migration/resolution reactions similar to those catalyzed by E. coli RuvABC, highlighting the preservation of this essential pathway in recombination and DNA repair from prokaryotes to mammals.
Assuntos
DNA Helicases , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli , Recombinação Genética , Animais , Proteínas de Bactérias/metabolismo , Fracionamento Celular , Linhagem Celular , Sistema Livre de Células , Cricetinae , Reparo do DNA , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Escherichia coli/química , Resolvases de Junção Holliday , Humanos , Substâncias Macromoleculares , Conformação de Ácido Nucleico , CoelhosRESUMO
RecA protein is essential for homologous recombination and the repair of DNA double-strand breaks in Escherichia coli. The protein binds DNA to form nucleoprotein filaments that promote joint molecule formation and strand exchange in vitro. RecA polymerises on ssDNA in the 5'-3' direction and catalyses strand exchange and branch migration with a 5'-3' polarity. It has been reported previously, using D-loop assays, in which ssDNA (containing a heterologous block at one end) invades supercoiled duplex DNA that 3'-homologous ends are reactive, whereas 5'-ends are inactive. This polarity bias was thought to be due to the polarity of RecA filament formation, which results in the 3'-ends being coated in RecA, whereas 5'-ends remain naked. Using a range of duplex substrates containing ssDNA tails of various lengths and polarities, we now demonstrate that when no heterologous block is imposed, 5'-ends are just as reactive as 3'-ends. Moreover, using short-tailed substrates, we find that 5'-ends form more stable D-loops than 3'-ends. This bias may be a consequence of the instability of short 3'-joints. With more physiological substrates containing long ssDNA tails, we find that RecA shows no intrinsic preference for 5' or 3'-ends and that both form D-loop complexes with high efficiency.
Assuntos
DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Conformação de Ácido Nucleico , Recombinases Rec A/metabolismo , DNA de Cadeia Simples/genética , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Genéticos , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Recombinação Genética/genética , Especificidade por SubstratoRESUMO
The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair.
Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA/genética , DNA/metabolismo , DNA/química , DNA/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Microscopia Eletrônica , Modelos Genéticos , Conformação de Ácido Nucleico , Ligação Proteica , Rad51 Recombinase , Recombinases Rec A/metabolismo , Recombinação Genética/genética , Proteína de Replicação A , Homologia de Sequência do Ácido NucleicoRESUMO
In mammalian cells, double-strand breaks in DNA can be repaired by nonhomologous end-joining (NHEJ), a process dependent upon Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV. Starting with HeLa cell-free extracts, which promote NHEJ in a reaction dependent upon all of these proteins, we have purified a novel factor that stimulates DNA end-joining in vitro. Using a combination of phosphorus NMR, mass spectroscopy, and strong anion exchange chromatography, we identify this factor as inositol hexakisphosphate (IP6). Purified IP6 is bound by DNA-PK and specifically stimulates DNA-PK-dependent end-joining in vitro. The involvement of inositol phosphate in DNA-PK-dependent NHEJ is of particular interest since the catalytic domain of DNA-PKcs is similar to that found in the phosphatidylinositol 3 (PI 3)-kinase family.
Assuntos
Reparo do DNA/fisiologia , DNA/metabolismo , Ácido Fítico/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fracionamento Celular , Sistema Livre de Células , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Teste de Complementação Genética , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Proteínas Nucleares , Ácido Fítico/análise , Ácido Fítico/isolamento & purificação , TrítioRESUMO
The Mycobacterium leprae RuvA homologue (MlRuvA) was over-expressed in Escherichia coli and purified to homogeneity. The DNA-binding specificity and the functional interactions of MlRuvA with E. coli RuvB and RuvC (EcRuvB and EcRuvC) were examined using synthetic Holliday junctions. MlRuvA bound specifically to Holliday junctions and produced similar band-shift patterns as EcRuvA. Moreover, MlRuvA formed functional DNA helicase and branch-migration enzymes with EcRuvB, although the heterologous enzyme had a lower efficiency. These results demonstrate that the RuvA homologue of M. leprae is a functional branch-migration subunit. Whereas MlRuvA promoted branch-migration in combination with EcRuvB, it was unable to stimulate branch-migration-dependent resolution in a RuvABC complex. The inability to stimulate RuvC was not due to its failure to form heterologous RuvABC complexes on junctions, since such complexes were detected by co-immunoprecipitation. Most likely, the stability of the heterologous RuvABC complex and, possibly, the interactions between RuvA and RuvC were impaired, as gel-shift experiments failed to show mixed MlRuvA-EcRuvC-junction complexes. These results demonstrate that branch-migration per se and the assembly of a RuvABC complex on the Holliday junction are insufficient for RuvAB-dependent resolution of the junction by RuvC, suggesting that specific and intimate interactions between all three proteins are required for the function of a RuvABC "resolvasome".
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Mycobacterium leprae/enzimologia , Recombinação Genética , Adenosina Trifosfatases/metabolismo , Cálcio/metabolismo , Cálcio/farmacologia , Cátions Bivalentes/metabolismo , Cátions Bivalentes/farmacologia , DNA/química , DNA/genética , DNA/metabolismo , DNA Helicases/metabolismo , Escherichia coli/genética , Substâncias Macromoleculares , Magnésio/metabolismo , Magnésio/farmacologia , Mycobacterium leprae/genética , Ligação Proteica/efeitos dos fármacos , Recombinação Genética/genética , Especificidade por SubstratoRESUMO
The human RAD52 protein, which exhibits a heptameric ring structure, has been shown to bind resected double strand breaks (DSBs), consistent with an early role in meiotic recombination and DSB repair. In this work, we show that RAD52 binds single-stranded and tailed duplex DNA molecules via precise interactions with the terminal base. When probed with hydroxyl radicals, ssDNA-RAD52 complexes exhibit a four-nucleotide repeat hypersensitivity pattern. This unique pattern is due to the interaction of RAD52 with either a 5' or a 3' terminus of the ssDNA, is sequence independent and is phased precisely from the terminal nucleotide. Hypersensitivity is observed over approximately 36 nucleotides, consistent with the length of DNA that is protected by RAD52 in nuclease protection assays. We propose that RAD52 binds DNA breaks via specific interactions with the terminal base, leading to the formation of a precisely organized ssDNA-RAD52 complex in which the DNA lies on an exposed surface of the protein. This protein-DNA arrangement may facilitate the DNA-DNA interactions necessary for RAD52-mediated annealing of complementary DNA strands.
Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sítios de Ligação , Dano ao DNA , Pegada de DNA , Reparo do DNA , Humanos , Radical Hidroxila , Modelos Moleculares , Ligação Proteica , Proteína Rad52 de Recombinação e Reparo de DNARESUMO
Cancer-causing mutations often arise from gross chromosomal rearrangements (GCRs) such as translocations, which involve genetic exchange between nonhomologous chromosomes. Here we show that murine Brca2 has an essential function in suppressing GCR formation after chromosome breakage. Cells that harbor truncated Brca2 spontaneously incur GCRs and genomic DNA breaks during division. They exhibit hypersensitivity to DNA damage by interstrand cross-linkers, which even at low doses trigger aberrant genetic exchange between nonhomologous chromosomes. Therefore, genetic instability in Brca2-deficient cells results from the mutagenic processing of spontaneous or induced DNA damage into gross chromosomal rearrangements, providing a mechanistic basis for cancer predisposition.
Assuntos
Aberrações Cromossômicas , Cromossomos/genética , Inativação Gênica , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Translocação Genética , Animais , Anexina A5/metabolismo , Proteína BRCA2 , Células Cultivadas , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Predisposição Genética para Doença , Marcação In Situ das Extremidades Cortadas , Cariotipagem , Fígado/embriologia , Camundongos , Mitomicina/farmacologia , Mutagênese , Rad51 Recombinase , Recombinação GenéticaRESUMO
The Escherichia coli RuvABC proteins process recombination intermediates during genetic recombination and recombinational repair. Although early biochemical studies indicated distinct RuvAB-mediated branch migration and RuvC-mediated Holliday junction resolution reactions, more recent studies have shown that the three proteins act together as a "resolvasome" complex. In this work we have used recombination intermediates made by RecA to determine whether the RuvAB proteins affect the sequence specificity of the RuvC resolvase. We find that RuvAB proteins do not alter significantly the site specificity of RuvC-dependent cleavage, although under certain conditions, they do affect the efficiency of cleavage at particular sites. The presence of RecA also influences cleavage at some sites. We also show that the RuvAB proteins act upon transient strand exchange intermediates made using substrates that have the opposite polarity of those preferred by RecA. Together, our results allow us to develop further a model for the recombinational repair of DNA lesions that lead to the formation of post-replication gaps during DNA replication. The novel features of this model are as follows: (i) the RuvABC resolvasome recognizes joints made by RecA; (ii) resolution by RuvABC occurs at specific sites containing the RuvC consensus cleavage sequence 5'-(A/T)TT downward arrow(G/C)-3'; and (iii) Holliday junction resolution often occurs close to the initiating gap without significant heteroduplex DNA formation.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Sequência Consenso , DNA Bacteriano/metabolismo , Eletroforese em Gel de Poliacrilamida , Substâncias Macromoleculares , Recombinases Rec A/metabolismoRESUMO
Bloom's syndrome (BS) is an autosomal recessive disorder associated with dwarfism, immunodeficiency, reduced fertility, and elevated levels of many types of cancer. BS cells show marked genomic instability; in particular, hyperrecombination between sister chromatids and homologous chromosomes. This instability is thought to result from defective processing of DNA replication intermediates. The gene mutated in BS, BLM, encodes a member of the RecQ family of DExH box DNA helicases, which also includes the Werner's syndrome gene product. We have investigated the mechanism by which BLM suppresses hyperrecombination. Here, we show that BLM selectively binds Holliday junctions in vitro and acts on recombination intermediates containing a Holliday junction to promote ATP-dependent branch migration. We present a model in which BLM disrupts potentially recombinogenic molecules that arise at sites of stalled replication forks. Our results have implications for the role of BLM as an anti-recombinase in the suppression of tumorigenesis.