A novel plant gene essential for meiosis is related to the human CtIP and the yeast COM1/SAE2 gene.
ABSTRACT: Obligatory homologous recombination (HR) is required for chiasma formation and chromosome segregation in meiosis I. Meiotic HR is initiated by DNA double-strand breaks (DSBs), generated by Spo11, a homologue of the archaebacterial topoisomerase subunit Top6A. In Saccharomyces cerevisiae, Rad50, Mre11 and Com1/Sae2 are essential to process an intermediate of the cleavage reaction consisting of Spo11 covalently linked to the 5' termini of DNA. While Rad50 and Mre11 also confer genome stability to vegetative cells and are well conserved in evolution, Com1/Sae2 was believed to be fungal-specific. Here, we identify COM1/SAE2 homologues in all eukaryotic kingdoms. Arabidopsis thaliana Com1/Sae2 mutants are sterile, accumulate AtSPO11-1 during meiotic prophase and fail to form AtRAd51 foci despite the presence of unrepaired DSBs. Furthermore, DNA fragmentation in AtCom1 is suppressed by eliminating AtSPO11-1. In addition, AtCOM1 is specifically required for mitomycin C resistance. Interestingly, we identified CtIP, an essential protein interacting with the DNA repair machinery, as the mammalian homologue of Com1/Sae2, with important implications for the molecular role of CtIP.
Project description:Meiotic recombination requires the formation of programmed Spo11-dependent DNA double strand breaks (DSBs). In Saccharomyces cerevisiae, the Sae2 protein and the Mre11-Rad50-Xrs2 complex are necessary to remove the covalently attached Spo11 protein from the DNA ends, which are then resected by so far unknown nucleases. Here, we demonstrate that phosphorylation of Sae2 Ser-267 by cyclin-dependent kinase 1 (Cdk1) is required to initiate meiotic DSB resection by allowing Spo11 removal from DSB ends. This finding suggests that Cdk1 activity is required for the processing of Spo11-induced DSBs, thus providing a mechanism for coordinating DSB resection with progression through meiotic prophase. Furthermore, the helicase Sgs1 and the nucleases Exo1 and Dna2 participate in lengthening the 5'-3' resection tracts during meiosis by controlling a step subsequent to Spo11 removal.
Project description:Meiotic programmed DNA double-strand break (DSB) repair is essential for crossing-over and viable gamete formation and requires removal of Spo11-oligonucleotide complexes from 5' ends (clipping) and their resection to generate invasive 3'-end single-stranded DNA (resection). Ctp1 (Com1, Sae2, CtIP homolog) acting with the Mre11-Rad50-Nbs1 (MRN) complex is required in both steps. We isolated multiple S. pombe ctp1 mutants deficient in clipping but proficient in resection during meiosis. Remarkably, all of the mutations clustered in or near the conserved CxxC or RHR motif in the C-terminal portion. The mutants tested, like ctp1?, were clipping-deficient by both genetic and physical assays-. But, unlike ctp1?, these mutants were recombination-proficient for Rec12 (Spo11 homolog)-independent break-repair and resection-proficient by physical assay. We conclude that the intracellular Ctp1 C-terminal portion is essential for clipping, while the N-terminal portion is sufficient for DSB end-resection. This conclusion agrees with purified human CtIP resection and endonuclease activities being independent. Our mutants provide intracellular evidence for separable functions of Ctp1. Some mutations truncate Ctp1 in the same region as one of the CtIP mutations linked to the Seckel and Jawad severe developmental syndromes, suggesting that these syndromes are caused by a lack of clipping at DSB ends that require repair.
Project description:Vertebrate CtIP, and its fission yeast (Ctp1), budding yeast (Sae2) and plant (Com1) orthologs have emerged as key regulatory molecules in cellular responses to DNA double strand breaks (DSBs). By modulating the nucleolytic 5'-3' resection activity of the Mre11/Rad50/Nbs1 (MRN) DSB repair processing and signaling complex, CtIP/Ctp1/Sae2/Com1 is integral to the channeling of DNA double strand breaks through DSB repair by homologous recombination (HR). Nearly two decades since its discovery, emerging new data are defining the molecular underpinnings for CtIP DSB repair regulatory activities. CtIP homologs are largely intrinsically unstructured proteins comprised of expanded regions of low complexity sequence, rather than defined folded domains typical of DNA damage metabolizing enzymes and nucleases. A compact structurally conserved N-terminus forms a functionally critical tetrameric helical dimer of dimers (THDD) region that bridges CtIP oligomers, and is flexibly appended to a conserved C-terminal Sae2-homology DNA binding and DSB repair pathway choice regulatory hub which influences nucleolytic activities of the MRN core nuclease complex. The emerging evidence from structural, biophysical, and biological studies converges on CtIP having functional roles in DSB repair that include: 1) dynamic DNA strand coordination through direct DNA binding and DNA bridging activities, 2) MRN nuclease complex cofactor functions that direct MRN endonucleolytic cleavage of protein-blocked DSB ends and 3) acting as a protein binding hub targeted by the cell cycle regulatory apparatus, which influences CtIP expression and activity via layers of post-translational modifications, protein-protein interactions and DNA binding.
Project description:The Schizosaccharomyces pombe nip1(+)/ctp1(+) gene was previously identified as an slr (synthetically lethal with rad2) mutant. Epistasis analysis indicated that Nip1/Ctp1 functions in Rhp51-dependent recombinational repair, together with the Rad32 (spMre11)-Rad50-Nbs1 complex, which plays important roles in the early steps of DNA double-strand break repair. Nip1/Ctp1 was phosphorylated in asynchronous, exponentially growing cells and further phosphorylated in response to bleomycin treatment. Overproduction of Nip1/Ctp1 suppressed the DNA repair defect of an nbs1-s10 mutant, which carries a mutation in the FHA phosphopeptide-binding domain of Nbs1, but not of an nbs1 null mutant. Meiotic DNA double-strand breaks accumulated in the nip1/ctp1 mutant. The DNA repair phenotypes and epistasis relationships of nip1/ctp1 are very similar to those of the Saccharomyces cerevisiae sae2/com1 mutant, suggesting that Nip1/Ctp1 is a functional homologue of Sae2/Com1, although the sequence similarity between the proteins is limited to the C-terminal region containing the RHR motif. We found that the RxxL and CxxC motifs are conserved in Schizosaccharomyces species and in vertebrate CtIP, originally identified as a cofactor of the transcriptional corepressor CtBP. However, these two motifs are not found in other fungi, including Saccharomyces and Aspergillus species. We propose that Nip1/Ctp1 is a functional counterpart of Sae2/Com1 and CtIP.
Project description:Faithful inheritance of genetic information through sexual reproduction relies on the formation of crossovers between homologous chromosomes during meiosis, which, in turn, relies on the formation and repair of numerous double-strand breaks (DSBs). As DSBs pose a potential threat to the genome, mechanisms that ensure timely and error-free DSB repair are crucial for successful meiosis. Here, we identify NBS-1, the Caenorhabditis elegans ortholog of the NBS1 (mutated in Nijmegen Breakage Syndrome) subunit of the conserved MRE11-RAD50-NBS1/Xrs2 (MRN) complex, as a key mediator of DSB repair via homologous recombination (HR) during meiosis. Loss of nbs-1 leads to severely reduced loading of recombinase RAD-51, ssDNA binding protein RPA, and pro-crossover factor COSA-1 during meiotic prophase progression; aggregated and fragmented chromosomes at the end of meiotic prophase; and 100% progeny lethality. These phenotypes reflect a role for NBS-1 in processing of meiotic DSBs for HR that is shared with its interacting partners MRE-11-RAD-50 and COM-1 (ortholog of Com1/Sae2/CtIP). Unexpectedly, in contrast to MRE-11 and RAD-50, NBS-1 is not required for meiotic DSB formation. Meiotic defects of the nbs-1 mutant are partially suppressed by abrogation of the nonhomologous end-joining (NHEJ) pathway, indicating a role for NBS-1 in antagonizing NHEJ during meiosis. Our data further reveal that NBS-1 and COM-1 play distinct roles in promoting HR and antagonizing NHEJ. We propose a model in which different components of the MRN-C complex work together to couple meiotic DSB formation with efficient and timely engagement of HR, thereby ensuring crossover formation and restoration of genome integrity before the meiotic divisions.
Project description:DNA ends exposed after introduction of double-strand breaks (DSBs) undergo 5'-3' nucleolytic degradation to generate single-stranded DNA, the substrate for binding by the Rad51 protein to initiate homologous recombination. This process is poorly understood in eukaryotes, but several factors have been implicated, including the Mre11 complex (Mre11-Rad50-Xrs2/NBS1), Sae2/CtIP/Ctp1 and Exo1. Here we demonstrate that yeast Exo1 nuclease and Sgs1 helicase function in alternative pathways for DSB processing. Novel, partially resected intermediates accumulate in a double mutant lacking Exo1 and Sgs1, which are poor substrates for homologous recombination. The early processing step that generates partly resected intermediates is dependent on Sae2. When Sae2 is absent, in addition to Exo1 and Sgs1, unprocessed DSBs accumulate and homology-dependent repair fails. These results suggest a two-step mechanism for DSB processing during homologous recombination. First, the Mre11 complex and Sae2 remove a small oligonucleotide(s) from the DNA ends to form an early intermediate. Second, Exo1 and/or Sgs1 rapidly process this intermediate to generate extensive tracts of single-stranded DNA that serve as substrate for Rad51.
Project description:Double-strand breaks (DSBs) in chromosomes are the most challenging type of DNA damage. The yeast and mammalian Mre11-Rad50-Xrs2/Nbs1 (MRX/N)-Sae2/Ctp1 complex catalyzes the resection of DSBs induced by secondary structures, chemical adducts or covalently-attached proteins. MRX/N also initiates two parallel DNA damage responses-checkpoint phosphorylation and global SUMOylation-to boost a cell's ability to repair DSBs. However, the molecular mechanism of this SUMO-mediated response is not completely known. In this study, we report that Saccharomyces cerevisiae Mre11 can non-covalently recruit the conjugated SUMO moieties, particularly the poly-SUMO chain. Mre11 has two evolutionarily-conserved SUMO-interacting motifs, Mre11(SIM1) and Mre11(SIM2), which reside on the outermost surface of Mre11. Mre11(SIM1) is indispensable for MRX assembly. Mre11(SIM2) non-covalently links MRX with the SUMO enzymes (E2/Ubc9 and E3/Siz2) to promote global SUMOylation of DNA repair proteins. Mre11(SIM2) acts independently of checkpoint phosphorylation. During meiosis, the mre11(SIM2) mutant, as for mre11S, rad50S and sae2?, allows initiation but not processing of Spo11-induced DSBs. Using MRX and DSB repair as a model, our work reveals a general principle in which the conjugated SUMO moieties non-covalently facilitate the assembly and functions of multi-subunit protein complexes.
Project description:The Mre11/Rad50/Nbs1 (MRN) complex is required for eukaryotic DNA double-strand break (DSB) repair and meiotic recombination. We cloned the Coprinus cinereus rad50 gene and showed that it corresponds to the complementation group previously named rad12, identified mutations in 15 rad50 alleles, and mapped two of the mutations onto molecular models of Rad50 structure. We found that C. cinereus rad50 and mre11 mutants arrest in meiosis and that this arrest is Spo11 dependent. In addition, some rad50 alleles form inducible, Spo11-dependent Rad51 foci and therefore must be forming meiotic DSBs. Thus, we think it likely that arrest in both mre11-1 and the collection of rad50 mutants is the result of unrepaired or improperly processed DSBs in the genome and that Rad50 and Mre11 are dispensable in C. cinereus for DSB formation, but required for appropriate DSB processing. We found that the ability of rad50 mutant strains to form Rad51 foci correlates with their ability to promote synaptonemal complex formation and with levels of stable meiotic pairing and that partial pairing, recombination initiation, and synapsis occur in the absence of wild-type Rad50 catalytic domains. Examination of single- and double-mutant strains showed that a spo11 mutation that prevents DSB formation enhances axial element (AE) formation for rad50-4, an allele predicted to encode a protein with intact hook region and hook-proximal coiled coils, but not for rad50-1, an allele predicted to encode a severely truncated protein, or for rad50-5, which encodes a protein whose hook-proximal coiled-coil region is disrupted. Therefore, Rad50 has an essential structural role in the formation of AEs, separate from the DSB-processing activity of the MRN complex.
Project description:Genome stability relies on faithful DNA repair both in mitosis and in meiosis. Here, we report on a Caenorhabditis elegans protein that we found to be homologous to the mammalian repair-related protein CtIP and to the budding yeast Com1/Sae2 recombination protein. A com-1 mutant displays normal meiotic chromosome pairing but forms irregular chromatin aggregates instead of diakinesis bivalents. While meiotic DNA double-strand breaks (DSBs) are formed, they appear to persist or undergo improper repair. Despite the presence of DSBs, the recombination protein RAD-51, which is known to associate with single-stranded DNA (ssDNA) flanking DSBs, does not localize to meiotic chromosomes in the com-1 mutant. Exposure of the mutant to gamma-radiation, however, induces RAD-51 foci, which suggests that the failure of RAD-51 to load is specific to meiotic (SPO-11-generated) DSBs. These results suggest that C. elegans COM-1 plays a role in the generation of ssDNA tails that can load RAD-51, invade homologous DNA tracts and thereby initiate recombination. Extrapolating from the worm homolog, we expect similar phenotypes for mutations in the mammalian tumor suppressor CtIP.
Project description:The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT(1)-BRCT(2) domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a bipartite MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.