RESUMEN
In meiosis, Dmc1 recombinase and the general recombinase Rad51 are responsible for pairing homologous chromosomes and exchanging strands. Fission yeast (Schizosaccharomyces pombe) Swi5-Sfr1 and Hop2-Mnd1 stimulate Dmc1-driven recombination, but the stimulation mechanism is unclear. Using single-molecule fluorescence resonance energy transfer (smFRET) and tethered particle motion (TPM) experiments, we showed that Hop2-Mnd1 and Swi5-Sfr1 individually enhance Dmc1 filament assembly on single-stranded DNA (ssDNA) and adding both proteins together allows further stimulation. FRET analysis showed that Hop2-Mnd1 enhances the binding rate of Dmc1 while Swi5-Sfr1 specifically reduces the dissociation rate during the nucleation, about 2-fold. In the presence of Hop2-Mnd1, the nucleation time of Dmc1 filaments shortens, and doubling the ss/double-stranded DNA (ss/dsDNA) junctions of DNA substrates reduces the nucleation times in half. Order of addition experiments confirmed that Hop2-Mnd1 binds on DNA to recruit and stimulate Dmc1 nucleation at the ss/dsDNA junction. Our studies directly support the molecular basis of how Hop2-Mnd1 and Swi5-Sfr1 act on different steps during the Dmc1 filament assembly. DNA binding of these accessory proteins and nucleation preferences of recombinases thus dictate how their regulation can take place.
Asunto(s)
Recombinasa Rad51 , Schizosaccharomyces , Proteínas de Ciclo Celular/metabolismo , ADN/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Meiosis , Recombinasa Rad51/metabolismo , Recombinasas/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5'-ended DNA strands at DSB ends, producing 3'-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Péptidos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Secuencia de Aminoácidos , Quinasa de la Caseína II/metabolismo , Secuencia Conservada , Roturas del ADN de Doble Cadena , FosforilaciónRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Recombinasa Rad51/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adenosina Trifosfatasas/metabolismo , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/fisiología , Inestabilidad Genómica , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/aislamiento & purificación , Proteínas de Schizosaccharomyces pombe/fisiologíaRESUMEN
Homologous recombination (HR) is a universal mechanism operating in somatic and germ-line cells, where it contributes to the maintenance of genome stability and ensures the faithful distribution of genetic material, respectively. The ability to identify and exchange the strands of two homologous DNA molecules lies at the heart of HR and is mediated by RecA-family recombinases. Dmc1 is a meiosis-specific RecA homolog in eukaryotes, playing a predominant role in meiotic HR. However, Dmc1 cannot function without its two major auxiliary factor complexes, Swi5-Sfr1 and Hop2-Mnd1. Through biochemical reconstitutions, we demonstrate that Swi5-Sfr1 and Hop2-Mnd1 make unique contributions to stimulate Dmc1-driven strand exchange in a synergistic manner. Mechanistically, Swi5-Sfr1 promotes establishment of the Dmc1 nucleoprotein filament, whereas Hop2-Mnd1 defines a critical, rate-limiting step in initiating strand exchange. Following execution of this function, we propose that Swi5-Sfr1 then promotes strand exchange with Hop2-Mnd1. Thus, our findings elucidate distinct yet complementary roles of two auxiliary factors in Dmc1-driven strand exchange, providing mechanistic insights into some of the most critical steps in meiotic HR.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga/fisiología , Recombinasa Rad51/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , ADN/metabolismo , Meiosis/fisiología , Rec A Recombinasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
The synaptonemal complex (SC) is a proteinaceous macromolecular assembly that forms during meiotic prophase I and mediates adhesion of paired homologous chromosomes along their entire lengths. Although prompt disassembly of the SC during exit from prophase I is a landmark event of meiosis, the underlying mechanism regulating SC destruction has remained elusive. Here, we show that DDK (Dbf4-dependent Cdc7 kinase) is central to SC destruction. Upon exit from prophase I, Dbf4, the regulatory subunit of DDK, directly associates with and is phosphorylated by the Polo-like kinase Cdc5. In parallel, upregulated CDK1 activity also targets Dbf4. An enhanced Dbf4-Cdc5 interaction pronounced phosphorylation of Dbf4 and accelerated SC destruction, while reduced/abolished Dbf4 phosphorylation hampered destruction of SC proteins. SC destruction relieved meiotic inhibition of the ubiquitous recombinase Rad51, suggesting that the mitotic recombination machinery is reactivated following prophase I exit to repair any persisting meiotic DNA double-strand breaks. Taken together, we propose that the concerted action of DDK, Polo-like kinase, and CDK1 promotes efficient SC destruction at the end of prophase I to ensure faithful inheritance of the genome.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Meiosis/fisiología , Proteínas Quinasas/metabolismo , Complejo Sinaptonémico/metabolismo , Fosforilación , Saccharomycetales/metabolismoRESUMEN
Here, we report the development of methodologies that enable genetic modification of a Basidiomycota yeast, Naganishia liquifaciens. The gene targeting method employs electroporation with PCR products flanked by an 80 bp sequence homologous to the target. The method, combined with a newly devised CRISPR-Cas9 system, routinely achieves 80% gene targeting efficiency. We further explored the genetic requirement for this homologous recombination (HR)-mediated gene targeting. The absence of Ku70, a major component of the non-homologous end joining (NHEJ) pathway of DNA double-strand break repair, almost completely eliminated inaccurate integration of the marker. Gene targeting with short homology (80 bp) was almost exclusively dependent on Rad52, an essential component of HR in the Ascomycota yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. By contrast, the RecA homolog Rad51, which performs homology search and strand exchange in HR, plays a relatively minor role in gene targeting, regardless of the homology length (80 bp or 1 kb). The absence of both Rad51 and Rad52, however, completely eliminated gene targeting. Unlike Ascomycota yeasts, the absence of Rad52 in N. liquefaciens conferred only mild sensitivity to ionizing radiation. These traits associated with the absence of Rad52 are reminiscent of findings in mice.
Asunto(s)
Basidiomycota/genética , Proteínas Fúngicas/metabolismo , Marcación de Gen , Recombinasa Rad51/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Sistemas CRISPR-Cas , Edición Génica , Prueba de Complementación Genética , Ingeniería Genética , Sitios Genéticos , Recombinación Homóloga , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Autoantígeno Ku/genética , Transformación GenéticaRESUMEN
The meiotic cell cycle provides a unique model to study the relationship between recombinational DNA repair and the cell cycle, since homologous recombination, induced by programmed DNA double-strand breaks (DSBs), is integrated as an essential step during meiosis. The pachytene checkpoint, which is situated towards the end of meiotic prophase I, coordinates homologous recombination and cell cycle progression, similar to the DNA damage checkpoint mechanisms operating in vegetative cells. However, there are a number of features unique to meiosis, making the system optimized for the purpose of meiosis. Our recent work highlights the involvement of three major cell cycle kinases, Dbf4-dependent Cdc7 kinase, Polo kinase and CDK, in coordinating homologous recombination and the meiotic cell cycle. In this review, we will discuss the unique interplay between meiotic cell cycle control and homologous recombination during meiosis I.
Asunto(s)
Daño del ADN/genética , Meiosis/genética , Profase Meiótica I/genética , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
During meiosis, homologous chromosomes pair at close proximity to form the synaptonemal complex (SC). This association is mediated by transverse filament proteins that hold the axes of homologous chromosomes together along their entire length. Transverse filament proteins are highly aggregative and can form an aberrant aggregate called the polycomplex that is unassociated with chromosomes. Here, we show that the Ecm11-Gmc2 complex is a novel SC component, functioning to facilitate assembly of the yeast transverse filament protein, Zip1. Ecm11 and Gmc2 initially localize to the synapsis initiation sites, then throughout the synapsed regions of paired homologous chromosomes. The absence of either Ecm11 or Gmc2 substantially compromises the chromosomal assembly of Zip1 as well as polycomplex formation, indicating that the complex is required for extensive Zip1 polymerization. We also show that Ecm11 is SUMOylated in a Gmc2-dependent manner. Remarkably, in the unSUMOylatable ecm11 mutant, assembly of chromosomal Zip1 remained compromised while polycomplex formation became frequent. We propose that the Ecm11-Gmc2 complex facilitates the assembly of Zip1 and that SUMOylation of Ecm11 is critical for ensuring chromosomal assembly of Zip1, thus suppressing polycomplex formation.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Fúngicas , Meiosis/genética , Proteínas de Saccharomyces cerevisiae , Complejo Sinaptonémico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrómero/genética , Centrómero/metabolismo , Emparejamiento Cromosómico/genética , Cromosomas Fúngicos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Sinaptonémico/genética , Complejo Sinaptonémico/metabolismoRESUMEN
The synaptonemal complex (SC) is a widely conserved structure that mediates the intimate alignment of homologous chromosomes during meiotic prophase and is required for proper homolog segregation at meiosis I. However, fundamental details of SC architecture and assembly remain poorly understood. The coiled-coil protein, Zip1, is the only component whose arrangement within the mature SC of budding yeast has been extensively characterized. It has been proposed that the Small Ubiquitin-like MOdifier, SUMO, plays a role in SC assembly by linking chromosome axes with Zip1's C termini. The role of SUMO in SC structure has not been directly tested, however, because cells lacking SUMO are inviable. Here, we provide direct evidence for SUMO's function in SC assembly. A meiotic smt3 reduction-of-function strain displays reduced sporulation, abnormal levels of crossover recombination, and diminished SC assembly. SC structures are nearly absent when induced at later meiotic time points in the smt3 reduction-of-function background. Using Structured Illumination Microscopy we furthermore determine the position of SUMO within budding yeast SC structure. In contrast to previous models that positioned SUMO near Zip1's C termini, we demonstrate that SUMO lies at the midline of SC central region proximal to Zip1's N termini, within a subdomain called the "central element". The recently identified SUMOylated SC component, Ecm11, also localizes to the SC central element. Finally, we show that SUMO, Ecm11, and even unSUMOylatable Ecm11 exhibit Zip1-like ongoing incorporation into previously established SCs during meiotic prophase and that the relative abundance of SUMO and Ecm11 correlates with Zip1's abundance within SCs of varying Zip1 content. We discuss a model in which central element proteins are core building blocks that stabilize the architecture of SC near Zip1's N termini, and where SUMOylation may occur subsequent to the incorporation of components like Ecm11 into an SC precursor structure.
Asunto(s)
Proteínas de Ciclo Celular/genética , Meiosis , Proteínas Nucleares/genética , Proteína SUMO-1/genética , Proteínas de Saccharomyces cerevisiae/genética , Complejo Sinaptonémico/genética , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Emparejamiento Cromosómico/genética , Segregación Cromosómica/genética , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/ultraestructura , Profase , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Sinaptonémico/ultraestructuraRESUMEN
The BRCA2 tumor suppressor plays a critical role in homologous recombination by regulating RAD51, the eukaryotic homologous recombinase. We identified the BRCA2 homolog in a Basidiomycota yeast, Naganishia liquefaciens BRCA2 homologs are found in many Basidiomycota species but not in Ascomycota species. Naganishia BRCA2 (Brh2, for BRCA2 homolog) is about one-third the size of human BRCA2. Brh2 carries three potential BRC repeats with two oligonucleotide/oligosaccharide-binding domains. The homolog of DSS1, a small acidic protein serving as an essential partner of BRCA2 was also identified. The yeast two-hybrid assay shows the interaction of Brh2 with both Rad51 and Dss1. Unlike human BRCA2, Brh2 is not required for normal cell growth, whereas loss of Dss1 results in slow growth. The loss of Brh2 caused pronounced sensitivity to UV and ionizing radiation, and their HR ability, as assayed by gene-targeting efficiency, is compromised. These phenotypes are indistinguishable from those of the rad51 mutant, and the rad51 brh2 double mutant. Naganishia Brh2 is likely the BRCA2 ortholog that functions as an indispensable auxiliary factor for Rad51.
Asunto(s)
Basidiomycota , Proteínas de Saccharomyces cerevisiae , Ustilago , Humanos , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al ADN/metabolismo , Recombinasa Rad51/genética , Reparación del ADN , Proteínas Fúngicas/metabolismo , Ustilago/genética , Ustilago/metabolismo , Basidiomycota/genética , Basidiomycota/metabolismo , Proteína BRCA2/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
During meiosis, recombination events that occur between homologous chromosomes help prepare the chromosome pairs for proper disjunction in meiosis I. The concurrent action of the Rad51 and Dmc1 recombinases is necessary for an interhomolog bias. Notably, the activity of Rad51 is tightly controlled, so as to minimize the use of the sister chromatid as recombination partner. We demonstrated recently that Hed1, a meiosis-specific protein in Saccharomyces cerevisiae, restricts the access of the recombinase accessory factor Rad54 to presynaptic filaments of Rad51. We now show that Hed1 undergoes self-association in a Rad51-dependent manner and binds ssDNA. We also find a strong stabilizing effect of Hed1 on the Rad51 presynaptic filament. Biochemical and genetic analyses of mutants indicate that these Hed1 attributes are germane for its recombination regulatory and Rad51 presynaptic filament stabilization functions. Our results shed light on the mechanism of action of Hed1 in meiotic recombination control.
Asunto(s)
Cromátides/metabolismo , Cromosomas Fúngicos/metabolismo , Meiosis/fisiología , Recombinasa Rad51/metabolismo , Recombinación Genética/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromátides/genética , Cromosomas Fúngicos/genética , ADN Helicasas , Enzimas Reparadoras del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Mutación , Recombinasa Rad51/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Homologous recombination (HR) is essential for meiosis in most sexually reproducing organisms, where it is induced upon entry into meiotic prophase. Meiotic HR is conducted by the collaborative effort of proteins responsible for DNA double-strand break repair and those produced specifically during meiosis. The Hop2-Mnd1 complex was originally identified as a meiosis-specific factor that is indispensable for successful meiosis in budding yeast. Later, it was found that Hop2-Mnd1 is conserved from yeasts to humans, playing essential roles in meiosis. Accumulating evidence suggests that Hop2-Mnd1 promotes RecA-like recombinases towards homology search/strand exchange. This review summarizes studies on the mechanism of the Hop2-Mnd1 complex in promoting HR and beyond.
Asunto(s)
Proteínas de Unión al ADN , Meiosis , Humanos , Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga , Reparación del ADN , Recombinasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMEN
Mating-type (P or M) of fission yeast Schizosaccharomyces pombe is determined by the transcriptionally active mat1 cassette and is switched by gene conversion using a donor, either mat2 or mat3, located in an adjacent heterochromatin region (mating-type switching; MTS). In the switching process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the action of two recombination enhancers, SRE2 promoting the use of mat2-P and SRE3 promoting the use of mat3-M, leading to replacement of the content of the expressed mat1 cassette. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1Δ. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant, we found that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in MTS in these mutants suggested that HULC and Set1C inhibit the use of the SRE3 recombination enhancer in M cells, thus favoring SRE2 and mat2-P. Consistent with this, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain.
Asunto(s)
Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Eucromatina/genética , Eucromatina/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Nucleosomas , Genes del Tipo Sexual de los Hongos/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Ligasas/genética , Ligasas/metabolismoRESUMEN
Homologous recombination (HR) is a universal phenomenon conserved from viruses to humans. The mechanisms of HR are essentially the same in humans and simple unicellular eukaryotes like yeast. Two highly diverged yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe, have proven exceptionally useful in understanding the fundamental mechanisms of eukaryotic HR by serving as a source for unique biological insights and also complementing each other. Here, we will review the features of S. pombe HR mechanisms in comparison to S. cerevisiae and other model organisms. Particular emphasis will be put on the biochemical characterization of HR mechanisms uncovered using S. pombe proteins.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Células Eucariotas , Recombinación Homóloga/genética , Humanos , Saccharomyces cerevisiae , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
DNA is the molecule that stores the chemical instructions necessary for life and its stability is therefore of the utmost importance. Despite this, DNA is damaged by both exogenous and endogenous factors at an alarming frequency. The most severe type of DNA damage is a double-strand break (DSB), in which a scission occurs in both strands of the double helix, effectively dividing a single normal chromosome into two pathological chromosomes. Homologous recombination (HR) is a universal DSB repair mechanism that solves this problem by identifying another region of the genome that shares high sequence similarity with the DSB site and using it as a template for repair. Rad51 possess the enzymatic activity that is essential for this repair but several auxiliary factors are required for Rad51 to fulfil its function. It is becoming increasingly clear that many HR factors are subjected to post-translational modification. Here, we review what is known about how these modifications affect HR. We first focus on cases where there is experimental evidence to support a function for the modification, then discuss speculative cases where a function can be inferred. Finally, we contemplate why such modifications might be necessary.
Asunto(s)
Procesamiento Proteico-Postraduccional , Reparación del ADN por Recombinación , Acetilación , Animales , Eucariontes , Humanos , Fosforilación , Recombinasa Rad51/metabolismo , Sumoilación , UbiquitinaciónRESUMEN
Homologous recombination (HR) is essential for maintaining genome stability. Although Rad51 is the key protein that drives HR, multiple auxiliary factors interact with Rad51 to potentiate its activity. Here, we present an interdisciplinary characterization of the interactions between Rad51 and these factors. Through structural analysis, we identified an evolutionarily conserved acidic patch of Rad51. The neutralization of this patch completely abolished recombinational DNA repair due to defects in the recruitment of Rad51 to DNA damage sites. This acidic patch was found to be important for the interaction with Rad55-Rad57 and essential for the interaction with Rad52. Furthermore, biochemical reconstitutions demonstrated that neutralization of this acidic patch also impaired the interaction with Rad54, indicating that a single motif is important for the interaction with multiple auxiliary factors. We propose that this patch is a fundamental motif that facilitates interactions with auxiliary factors and is therefore essential for recombinational DNA repair.
The DNA molecule contains the chemical instructions necessary for life. Its physical integrity is therefore vital, yet it is also under constant threat from external and internal factors. As a result, organisms have evolved an arsenal of mechanisms to repair damaged DNA. For instance, when the two complementary strands that form the DNA molecule are broken at the same location, the cell triggers a mechanism known as homologous recombination. A protein known as Rad51 orchestrates this process, helped by an array of other proteins that include Rad55-Rad57, Rad52, and Rad54. These physically bind to Rad51 and activate it in different ways. However, exactly how these interactions take place remained unclear. To find out more, Afshar et al. examined models of the structure of Rad51, revealing that three of the protein's building blocks create a prominent, negatively charged patch that could be important for DNA repair. Yeast cells were then genetically manipulated to produce a modified version of Rad51 in which the three building blocks were neutralised. These organisms were unable to repair their DNA. Further biochemical tests showed that the modified protein could no longer attach well to Rad55-Rad57 or Rad54, and could not stick to Rad52 at all. In fact, without its negatively charged patch, Rad51 could not find the ends of broken DNA strands, a process which is normally aided by Rad55-Rad57 and Rad52. Taken together, these results suggest that the helper proteins all interact with Rad51 in the same place, even though they play different roles. Faulty DNA repair processes have been linked to devastating consequences such as cell death or cancer. Understanding the details of DNA repair in yeast can serve as a template for research in more complex organisms, opening the possibility of applications for human health.
Asunto(s)
Recombinación Homóloga/genética , Recombinasa Rad51/genética , Reparación del ADN por Recombinación/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Recombinasa Rad51/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Genetic studies in budding and fission yeasts have provided evidence that Rdh54, a Swi2/Snf2-like factor, synergizes with the Dmc1 recombinase to mediate inter-homologue recombination during meiosis. Rdh54 associates with Dmc1 in the yeast two-hybrid assay, but whether the Rdh54-Dmc1 interaction is direct and the manner in which these two recombination factors may functionally co-operate to accomplish their biological task have not yet been defined. Here, using purified Schizosaccharomyces pombe proteins, we demonstrate complex formation between Rdh54 and Dmc1 and enhancement of the recombinase activity of Dmc1 by Rdh54. Consistent with published cytological and chromatin immunoprecipitation data that implicate Rdh54 in preventing the non-specific association of Dmc1 with chromatin, we show here that Rdh54 mediates the efficient removal of Dmc1 from dsDNA. These functional attributes of Rdh54 are reliant on its ATPase function. The results presented herein provide valuable information concerning the Rdh54-Dmc1 protein pair that is germane for understanding their role in meiotic recombination. The biochemical systems established in this study should be useful for the continuing dissection of the action mechanism of Rdh54 and Dmc1.
Asunto(s)
Meiosis , Recombinasas/metabolismo , Recombinación Genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Adenosina Trifosfatasas/metabolismo , ADN de Hongos/metabolismo , Conformación de Ácido Nucleico , Unión Proteica , Recombinasas/aislamiento & purificación , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/aislamiento & purificaciónRESUMEN
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.
RESUMEN
Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. We present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a conserved auxiliary factor. Two distinct sites within the intrinsically disordered N-terminus of Sfr1 (Sfr1N) were found to cooperatively bind Rad51. Deletion of this domain impaired Rad51 stimulation in vitro and rendered cells sensitive to DNA damage. By contrast, amino acid-substitution mutants, which had comparable biochemical defects, could promote DNA repair, suggesting that Sfr1N has another role in addition to Rad51 binding. Unexpectedly, the DNA repair observed in these mutants was dependent on Rad55-Rad57, another auxiliary factor complex hitherto thought to function independently of Swi5-Sfr1. When combined with the finding that they form a higher-order complex, our results imply that Swi5-Sfr1 and Rad55-Rad57 can collaboratively stimulate Rad51 in Schizosaccharomyces pombe.
The DNA within cells contains the instructions necessary for life and it must be carefully maintained. DNA is constantly being damaged by radiation and other factors so cells have evolved an arsenal of mechanisms that repair this damage. An enzyme called Rad51 drives one such DNA repair process known as homologous recombination. A pair of regulatory proteins known as the Swi5-Sfr1 complex binds to Rad51 and activates it. The complex can be thought of as containing two modules with distinct roles: one comprising the first half of the Sfr1 protein and that is capable of binding to Rad51, and a second consisting of the rest of Sfr1 bound to Swi5, which is responsible for activating Rad51. Here, Argunhan, Sakakura et al. used genetic and biochemical approaches to study how this first module, known as "Sfr1N", interacts with Rad51 in a microbe known as fission yeast. The experiments showed that both modules of Swi5-Sfr1 were important for Rad51 to drive homologous recombination. Swi5-Sfr1 complexes carrying mutations in the region of Sfr1N that binds to Rad51 were unable to activate Rad51 in a test tube. However, fission yeast cells containing the same mutations were able to repair their DNA without problems. This was due to the presence of another pair of proteins known as the Rad55-Rad57 complex that also bound to Swi5-Sfr1. The findings of Argunhan, Sakakura et al. suggest that the Swi5-Sfr1 and Rad55-Rad57 complexes work together to activate Rad51. Many genetically inherited diseases and cancers have been linked to mutations in DNA repair proteins. The fundamental mechanisms of DNA repair are very similar from yeast to humans and other animals, therefore, understanding the details of DNA repair in yeast may ultimately benefit human health in the future.
Asunto(s)
Daño del ADN/fisiología , Reparación del ADN/fisiología , Recombinasa Rad51/metabolismo , Schizosaccharomyces/metabolismo , Escherichia coli , Regulación Fúngica de la Expresión Génica , Espectroscopía de Resonancia Magnética , Unión Proteica , Dominios Proteicos , Recombinasa Rad51/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
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.