RESUMO
Two newly duplicated copies of genomic DNA are held together by the ring-shaped cohesin complex to ensure faithful inheritance of the genome during cell division1-3. Cohesin mediates sister chromatid cohesion by topologically entrapping two sister DNAs during DNA replication4,5, but how cohesion is established at the replication fork is poorly understood. Here, we studied the interplay between cohesin and replication by reconstituting a functional replisome using purified proteins. Once DNA is encircled before replication, the cohesin ring accommodates replication in its entirety, from initiation to termination, leading to topological capture of newly synthesized DNA. This suggests that topological cohesin loading is a critical molecular prerequisite to cope with replication. Paradoxically, topological loading per se is highly rate limiting and hardly occurs under the replication-competent physiological salt concentration. This inconsistency is resolved by the replisome-associated cohesion establishment factors Chl1 helicase and Ctf4 (refs. 6,7), which promote cohesin loading specifically during continuing replication. Accordingly, we found that bubble DNA, which mimics the state of DNA unwinding, induces topological cohesin loading and this is further promoted by Chl1. Thus, we propose that cohesin converts the initial electrostatic DNA-binding mode to a topological embrace when it encounters unwound DNA structures driven by enzymatic activities including replication. Together, our results show how cohesin initially responds to replication, and provide a molecular model for the establishment of sister chromatid cohesion.
Assuntos
Coesinas , Replicação do DNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cromátides/metabolismo , Coesinas/metabolismo , DNA Fúngico/biossíntese , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Eletricidade EstáticaRESUMO
OBJECTIVES: Sarcopenia has been reported as a prognostic risk factor in patients with gastrointestinal (GI) and hepatobiliary pancreatic (HBP) cancers. This study aimed to investigate whether the loss of muscle mass or strength is a stronger prognostic factor, and explore the cutoff values of skeletal muscle mass index (SMI) and handgrip strength (HGS) based on the survival outcome in patients with GI and HBP cancers. METHODS: A total of 480 elderly patients with primary GI and HBP cancers who underwent their first resection surgery were analyzed retrospectively. Patients were divided into four groups: Appropriate SMI and HGS, low SMI alone, low HGS alone, and low SMI and HGS. Low SMI was derived from a bioelectrical impedance analysis, and low HGS was defined according to the Asian Working Group for Sarcopenia 2019 criteria. RESULTS: The multivariate analysis showed that low SMI was a significant risk factor for mortality in men only, but low HGS was significant in both sexes. From the multivariate analysis of the four groups, low HGS alone and low SMI and HGS showed a significantly higher hazard ratio than appropriate SMI and HGS in both sexes. An SMI of 7.21 kg/m2 and HGS of 28 kg were obtained as cutoff values based on the 3-y survival outcomes in men. CONCLUSIONS: Low muscle strength was a stronger prognostic factor than low muscle mass. Therefore, measuring muscle strength in all patients is essential.
Assuntos
Neoplasias Pancreáticas , Sarcopenia , Masculino , Feminino , Humanos , Idoso , Sarcopenia/etiologia , Sarcopenia/patologia , Força da Mão/fisiologia , Prognóstico , Estudos Retrospectivos , Força Muscular , Músculo Esquelético/patologia , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/cirurgia , Neoplasias Pancreáticas/patologiaRESUMO
The Global Leadership Initiative on Malnutrition (GLIM) criteria recommends using race- and sex-adjusted cutoff values for reduced muscle mass (RMM), but the only cutoff values available for Asians are the skeletal muscle mass index (SMI) established by the Asian Working Group for Sarcopenia (AWGS). This retrospective study aimed to develop and validate cutoff values for the fat-free mass index (FFMI) and arm circumference (AC) of Asians, and to investigate the association between GLIM malnutrition and prognosis. A total of 660 patients with primary gastrointestinal (GI) and hepatobiliary-pancreatic (HBP) cancers who underwent their first resection surgery were recruited and randomly divided into development and validation groups. The FFMI and AC cutoff values were calculated by receiver operating characteristic curve analysis for the AWGS SMI as the gold standard. The cutoff values for each RMM were used to diagnose malnutrition on the basis of GLIM criteria, and the survival rates were compared. The optimal FFMI cutoff values for RMM were 17 kg/m2 for men and 15 kg/m2 for women, and for AC were 27 cm for men and 25 cm for women. In the validation group, the accuracy of the FFMI and AC cutoff values to discriminate RMM were 85.2% and 68.8%, respectively. Using any of the three measures of RMM, overall survival rates were significantly lower in the GLIM malnutrition group. In conclusion, the cutoff values for the FFMI and AC in this study could discriminate RMM, and GLIM malnutrition using these cutoff values was associated with decreased survival.
Assuntos
Desnutrição , Neoplasias Pancreáticas , Feminino , Humanos , Liderança , Masculino , Desnutrição/diagnóstico , Músculos , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/cirurgia , Estudos RetrospectivosRESUMO
Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenosina Trifosfatases/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/fisiologia , Instabilidade Genômica , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/fisiologiaRESUMO
Cohesin, a critical mediator of genome organization including sister chromatid cohesion, is a ring-shaped multi-subunit ATPase that topologically embraces DNA. Its loading and function on chromosomes require the Scc2-Scc4 loader. Using biochemical reconstitution, we show here that the ability of the loader to bind DNA plays a critical role in promoting cohesin loading. Two distinct sites within the Mis4Scc2 subunit are found to cooperatively bind DNA. Mis4Scc2 initially forms a tertiary complex with cohesin on DNA and promotes subsequent topological DNA entrapment by cohesin through its DNA binding activity, a process that requires an additional DNA binding surface provided by Psm3Smc3, the ATPase domain of cohesin. Furthermore, we show that mutations in the two DNA binding sites of Mis4 impair the chromosomal loading of cohesin. These observations demonstrate the physiological importance of DNA binding by the loader and provide mechanistic insights into the process of topological cohesin loading.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , DNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Cromossômicas não Histona , Segregação de Cromossomos , CoesinasRESUMO
The draft genome sequence of the deep-sea yeast Naganishia liquefaciens strain N6, isolated from the Japan Trench, is reported here. This strain was previously classified into a Cryptococcus clade. Phylogenetic analysis using the presented sequence suggests that strain N6 is in the clade of the genus Naganishia.
RESUMO
During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.
Assuntos
DNA/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/genética , DNA/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , DNA de Cadeia Simples/genética , Recombinação Homóloga/genética , Recombinação Homóloga/fisiologia , Humanos , Mutação/genética , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Estrutura Secundária de Proteína , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genéticaRESUMO
Eukaryotic Rad51 protein is essential for homologous-recombination repair of DNA double-strand breaks. Rad51 recombinases first assemble onto single-stranded DNA to form a nucleoprotein filament, required for function in homology pairing and strand exchange. This filament assembly is the first regulation step in homologous recombination. Rad51 nucleation is kinetically slow, and several accessory factors have been identified to regulate this step. Swi5-Sfr1 (S5S1) stimulates Rad51-mediated homologous recombination by stabilizing Rad51 nucleoprotein filaments, but the mechanism of stabilization is unclear. We used single-molecule tethered particle motion experiments to show that mouse S5S1 (mS5S1) efficiently stimulates mouse RAD51 (mRAD51) nucleus formation and inhibits mRAD51 dissociation from filaments. We also used single-molecule fluorescence resonance energy transfer experiments to show that mS5S1 promotes stable nucleus formation by specifically preventing mRAD51 dissociation. This leads to a reduction of nucleation size from three mRAD51 to two mRAD51 molecules in the presence of mS5S1. Compared with mRAD51, fission yeast Rad51 (SpRad51) exhibits fast nucleation but quickly dissociates from the filament. SpS5S1 specifically reduces SpRad51 disassembly to maintain a stable filament. These results clearly demonstrate the conserved function of S5S1 by primarily stabilizing Rad51 on DNA, allowing both the formation of the stable nucleus and the maintenance of filament length.
Assuntos
Proteínas Nucleares/metabolismo , Rad51 Recombinase/metabolismo , Animais , DNA , Recombinação Homóloga/fisiologia , Camundongos , Nucleoproteínas/metabolismo , Schizosaccharomyces/metabolismoRESUMO
The human skin microbiome can vary over time, and inter-individual variability of the microbiome is greater than the temporal variability within an individual. The skin microbiome has become a useful tool to identify individuals, and one type of personal identification using the skin microbiome has been reported in a community of less than 20 individuals. However, identification of individuals based on the skin microbiome has shown low accuracy in communities larger than 80 individuals. Here, we developed a new approach for personal identification, which considers that minor taxa are one of the important factors for distinguishing between individuals. We originally established a human skin microbiome for 66 samples from 11 individuals over two years (33 samples each year). Our method could classify individuals with 85% accuracy beyond a one-year sampling period. Moreover, we applied our method to 837 publicly available skin microbiome samples from 89 individuals and succeeded in identifying individuals with 78% accuracy. In short, our results investigate that (i) our new personal identification method worked well with two different communities (our data: 11 individuals; public data: 89 individuals) using the skin microbiome, (ii) defining the personal skin microbiome requires samples from several time points, (iii) inclusion of minor skin taxa strongly contributes to the effectiveness of personal identification.
Assuntos
Classificação , Microbiota , Registros , Pele/microbiologia , Adulto , Feminino , Humanos , Concentração de Íons de Hidrogênio , Masculino , Sebo/metabolismo , Sebo/microbiologia , Pele/química , Pele/metabolismo , Adulto JovemRESUMO
The ring-shaped structural maintenance of chromosome (SMC) complexes are multi-subunit ATPases that topologically encircle DNA. SMC rings make vital contributions to numerous chromosomal functions, including mitotic chromosome condensation, sister chromatid cohesion, DNA repair, and transcriptional regulation. They are thought to do so by establishing interactions between more than one DNA. Here, we demonstrate DNA-DNA tethering by the purified fission yeast cohesin complex. DNA-bound cohesin efficiently and topologically captures a second DNA, but only if that is single-stranded DNA (ssDNA). Like initial double-stranded DNA (dsDNA) embrace, second ssDNA capture is ATP-dependent, and it strictly requires the cohesin loader complex. Second-ssDNA capture is relatively labile but is converted into stable dsDNA-dsDNA cohesion through DNA synthesis. Our study illustrates second-DNA capture by an SMC complex and provides a molecular model for the establishment of sister chromatid cohesion.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Cromátides/metabolismo , Replicação do DNA , Saccharomyces cerevisiae , Schizosaccharomyces , CoesinasRESUMO
Previous researches suggest that addressing alexithymia and emotional dysregulation in patients with alcohol and drug addiction is important. However, interventions focused on emotional problems in Substance Use Disorder (SUD) patients are rare in Japanese hospital settings. In 2014, we developed and implemented an intervention called the Serigaya Collaboration for Open heart Project (SCOP) for those SUD inpatients who have difficulty recognizing and expressing their emotions. The SCOP consists of a series of emotion-focused group psychotherapies provided by psychologist, occupational therapist, and nurse. The purpose of this preliminary study is to assess the efficacy of.SCOP in SUD patients. A total of 65 inpatients (37 alcohol and 28 drug use disorder) participated in the research. Thirty-two patients consented to receive the SCOP intervention concurrently with treatment as usual (TAU), while the rest of the patients (n = 33) received only TAU. TAU consists of cognitive-behavioral group therapy, psychoeducational group sessions, occupational therapy, and on-site self-help group attendances. Based on medical records, we assessed retrospectively the 6 months prognosis after discharge. A comparison was made between the SCOP and TAU groups in terms of treatment retention, attendance at self-help group meetings, -and alcohol or drug relapse. No dif- ference in post-discharge treatment retention was observed between the groups. The SCOP group had more self-help group attendees and fewer patients with alcohol or drug relapse than the TAU group. Although our present study is retrospective in design, our preliminary findings suggest that the SCOP intervention which focuses on emotions may be effective in encouraging SUD patients to join self-help groups, and helped prevent alcohol or drug relapses.
Assuntos
Emoções , Psicoterapia de Grupo , Transtornos Relacionados ao Uso de Substâncias/terapia , Adulto , Feminino , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Pacientes Ambulatoriais , Transtornos Relacionados ao Uso de Substâncias/psicologiaRESUMO
Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCFFbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated.
Assuntos
DNA Helicases/genética , Recombinação Homóloga/genética , Rad51 Recombinase/biossíntese , Proteínas de Schizosaccharomyces pombe/biossíntese , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Citoesqueleto/genética , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Mutação , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Rad51 Recombinase/genética , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Both ubiquitously expressed Rad51 and meiosis-specific Dmc1 are required for crossover production during meiotic recombination. The budding yeast Rad52 and its fission yeast ortholog, Rad22, are "mediators;" i.e., they help load Rad51 onto ssDNA coated with replication protein A (RPA). Here we show that the Swi5-Sfr1 complex from fission yeast is both a mediator that loads Dmc1 onto ssDNA and a direct "activator" of DNA strand exchange by Dmc1. In stark contrast, Rad22 inhibits Dmc1 action by competing for its binding to RPA-coated ssDNA. Thus, Rad22 plays dual roles in regulating meiotic recombination: activating Rad51 and inhibiting Dmc1.
Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recombinases/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Trifosfato de Adenosina/metabolismo , Troca Genética/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Recombinação Homóloga , Meiose , Ligação Proteica , Estabilidade Proteica , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The human glycine-N-acyltransferase (hGLYAT) gene and two related-genes (GLYATL1 and GLYATL2) were isolated. Human GLYAT, GLYATL1, and GLYATL2 cDNAs were isolated and shown to encode polypeptides of 295, 302, and 294 amino acids, respectively. GLYAT catalyzes glycine-N-acyltransfer reaction with benzoyl-CoA acting as a typical aralkyl transferase, while GLYATL1 catalyzed glutamine-N-acyltransfer reaction with phenylacetyl-CoA as an arylacetyl transferase. GLYAT was shown to be expressed specifically in the liver and kidney, and the cellular localization of GLYAT protein was restricted to the mitochondria. Interestingly, labeling using highly affinity purified anti-GLYAT antibody revealed that GLYAT expression was suppressed in all hepatocellular carcinomas, but not in other liver diseases. hGLYAT repression in cancerous cells in the liver was controlled at the transcriptional level. hGLYAT is a good candidate as a novel marker of hepatocellular carcinoma and may be a key molecule in the transition between differentiation and carcinogenesis of liver cells.
Assuntos
Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Carcinoma Hepatocelular/enzimologia , Neoplasias Hepáticas/enzimologia , Acil Coenzima A/metabolismo , Aciltransferases/genética , Sequência de Aminoácidos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Mitocôndrias Hepáticas/enzimologia , Dados de Sequência MolecularRESUMO
Condensin is required for chromosome dynamics and diverse DNA metabolism. How condensin works, however, is not well understood. Condensin contains two structural maintenance of chromosomes (SMC) subunits with the terminal globular domains connected to coiled-coil that is interrupted by the central hinge. Heterotrimeric non-SMC subunits regulate SMC. We identified a novel fission yeast SMC hinge mutant, cut14-Y1, which displayed defects in DNA damage repair and chromosome segregation. It contains an amino acid substitution at a conserved hinge residue of Cut14/SMC2, resulting in diminished DNA binding and annealing. A replication protein A mutant, ssb1-418, greatly alleviated the repair and mitotic defects of cut14-Y1. Ssb1 protein formed nucleolar foci in cut14-Y1 cells, but the number of foci was diminished in cut14-Y1 ssb1-418 double mutants. Consistent with the above results, Ssb1 protein bound to single-strand DNA was removed by condensin or the SMC dimer through DNA reannealing in vitro. Similarly, RNA hybridized to DNA may be removed by the SMC dimer. Thus, condensin may wind up DNA strands to unload chromosomal components after DNA repair and prior to mitosis. We show that 16 suppressor mutations of cut14-Y1 were all mapped within the hinge domain, which surrounded the original L543 mutation site.
Assuntos
Adenosina Trifosfatases/química , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Complexos Multiproteicos/química , Proteína de Replicação A/química , Proteínas de Schizosaccharomyces pombe/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Mapeamento Cromossômico , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Dano ao DNA , Reparo do DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Fúngicos , Interfase , Mitose , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Homologia de Sequência de AminoácidosRESUMO
In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog) and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog)-mediated homologous recombination (HR) and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A (RPA), which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA (ssDNA) precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1-mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA.
Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Reparo do DNA , Proteína de Replicação A/metabolismoRESUMO
Holliday junctions (HJs) are key intermediates in homologous recombination and are especially important for the production of crossover recombinants. Bacterial RecA family proteins promote the formation and branch migration of HJs in vitro by catalysing a reciprocal DNA-strand exchange reaction between two duplex DNA molecules, one of which contains a single-stranded DNA region that is essential for initial nucleoprotein filament formation. This activity has been reported only for prokaryotic RecA family recombinases, although eukaryotic homologues are also essential for HJ production in vivo. Here we show that fission yeast (Rhp51) and human (hRad51) RecA homologues promote duplex-duplex DNA-strand exchange in vitro. As with RecA, a HJ is formed between the two duplex DNA molecules, and reciprocal strand exchange proceeds through branch migration of the HJ. In contrast to RecA, however, strand exchange mediated by eukaryotic recombinases proceeds in the 3'-->5' direction relative to the single-stranded DNA region of the substrate DNA. The opposite polarity of Rhp51 makes it especially suitable for the repair of DNA double-strand breaks, whose repair is initiated at the processed ends of breaks that have protruding 3' termini.
Assuntos
DNA Cruciforme/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Adenina/metabolismo , DNA Cruciforme/química , DNA Cruciforme/genética , Humanos , Recombinases Rec A/metabolismoRESUMO
The Schizosaccharomyces pombe Swi5 protein forms two distinct protein complexes, Swi5-Sfr1 and Swi5-Swi2, each of which plays an important role in the related but functionally distinct processes of homologous recombination and mating-type switching, respectively. The Swi5-Sfr1 mediator complex has been shown to associate with the two RecA-like recombinases, Rhp51 (spRad51) and Dmc1, and to stimulate in vitro DNA strand exchange reactions mediated by these proteins. Genetic analysis indicates that Swi5-Sfr1 works independently of another mediator complex, Rhp55-Rhp57, during Rhp51-dependent recombinational repair. In addition, mutations affecting the two mediators generate distinct repair spectra of HO endonuclease-induced DNA double strand breaks, suggesting that these recombination mediators differently regulate recombination outcomes in an independent manner.
Assuntos
DNA Fúngico/genética , Recombinação Genética/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/metabolismo , Reparo do DNA , Genes Fúngicos , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Several accessory proteins referred to as mediators are required for the full activity of the Rad51 (Rhp51 in fission yeast) recombinase. In this study, we analyzed in vivo functions of the recently discovered Swi5/Sfr1 complex from fission yeast. In normally growing cells, the Swi5-GFP protein localizes to the nucleus, where it forms a diffuse nuclear staining pattern with a few distinct foci. These spontaneous foci do not form in swi2Delta mutants. Upon UV irradiation, Swi5 focus formation is induced in swi2Delta mutants, a response that depends on Sfr1 function, and Sfr1 also forms foci that colocalize with damage-induced Rhp51 foci. The number of UV-induced Rhp51 foci is partially reduced in swi5Delta and rhp57Delta mutants and completely abolished in an swi5Delta rhp57Delta double mutant. An assay for products generated by HO endonuclease-induced DNA double-strand breaks (DSBs) reveals that Rhp51 and Rhp57, but not Swi5/Sfr1, are essential for crossover production. These results suggest that Swi5/Sfr1 functions as an Rhp51 mediator but processes DSBs in a manner different from that of the Rhp55/57 mediator.
Assuntos
Recombinação Genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA , Reparo do DNA/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Heterocromatina/metabolismo , Microscopia de Fluorescência , Ligação Proteica , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/genética , Raios UltravioletaRESUMO
Nucleoprotein filaments made up of Rad51 or Dmc1 recombinases, the core structures of recombination, engage in ATP-dependent DNA-strand exchange. The ability of recombinases to form filaments is enhanced by recombination factors termed 'mediators'. Here, we show that the Schizosaccharomyces pombe Swi5-Sfr1 complex, a conserved eukaryotic protein complex, at substoichiometric concentrations stimulates strand exchange mediated by Rhp51 (the S. pombe Rad51 homolog) and Dmc1 on long DNA substrates. Reactions mediated by both recombinases are completely dependent on Swi5-Sfr1, replication protein A (RPA) and ATP, although RPA inhibits the reaction when it is incubated with single-stranded DNA (ssDNA) before the recombinase. The Swi5-Sfr1 complex overcomes, at least partly, the inhibitory effect of RPA, representing a novel class of mediator. Notably, the Swi5-Sfr1 complex preferentially stimulates the ssDNA-dependent ATPase activity of Rhp51, and it increases the amounts of Dmc1 bound to ssDNA.