PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (1320751)

Clipboard (0)
None

Related Articles

1.  Structural Maintenance of Chromosomes (SMC) Proteins Promote Homolog-Independent Recombination Repair in Meiosis Crucial for Germ Cell Genomic Stability 
PLoS Genetics  2010;6(7):e1001028.
In meiosis, programmed DNA breaks repaired by homologous recombination (HR) can be processed into inter-homolog crossovers that promote the accurate segregation of chromosomes. In general, more programmed DNA double-strand breaks (DSBs) are formed than the number of inter-homolog crossovers, and the excess DSBs must be repaired to maintain genomic stability. Sister-chromatid (inter-sister) recombination is postulated to be important for the completion of meiotic DSB repair. However, this hypothesis is difficult to test because of limited experimental means to disrupt inter-sister and not inter-homolog HR in meiosis. We find that the conserved Structural Maintenance of Chromosomes (SMC) 5 and 6 proteins in Caenorhabditis elegans are required for the successful completion of meiotic homologous recombination repair, yet they appeared to be dispensable for accurate chromosome segregation in meiosis. Mutations in the smc-5 and smc-6 genes induced chromosome fragments and dismorphology. Chromosome fragments associated with HR defects have only been reported in mutants, which have disrupted inter-homolog crossover. Surprisingly, the smc-5 and smc-6 mutations did not disrupt the formation of chiasmata, the cytologically visible linkages between homologous chromosomes formed from meiotic inter-homolog crossovers. The mutant fragmentation defect appeared to be preferentially enhanced by the disruptions of inter-homolog recombination but not by the disruptions of inter-sister recombination. Based on these findings, we propose that the C. elegans SMC-5/6 proteins are required in meiosis for the processing of homolog-independent, presumably sister-chromatid-mediated, recombination repair. Together, these results demonstrate that the successful completion of homolog-independent recombination is crucial for germ cell genomic stability.
Author Summary
Sperm and oocytes are essential for the faithful transmission of genetic information during sexual reproduction. As germ cells mature into sperm and oocytes, DNA double-strand breaks (DSBs) are deliberately created on each chromosome and a subset of DSBs is repaired to form meiotic crossovers between homologous chromosomes. Because germ cells must undergo this programmed process of deliberate DNA damage and repair, identifying repair factors active in germ cells and determining the requirement of their functions in meiotic DSB repair are important first steps in understanding infertility and developmental disorders caused by defective sperm and oocytes. In this manuscript, we find that the evolutionarily conserved SMC-5 and SMC-6 proteins fulfill a critical role in preserving genomic stability in germ cells in C. elegans. Our findings further describe the genetic mechanisms by which the C. elegans SMC-5/6 proteins function in meiotic DSB repair. These data reveal that inter-sister homologous recombination, a repair mechanism thought to function as a back-up repair method in meiosis, serves a more significant role in normal meiosis than was previously appreciated.
doi:10.1371/journal.pgen.1001028
PMCID: PMC2908675  PMID: 20661436
2.  Importance of Polη for Damage-Induced Cohesion Reveals Differential Regulation of Cohesion Establishment at the Break Site and Genome-Wide 
PLoS Genetics  2013;9(1):e1003158.
Genome integrity depends on correct chromosome segregation, which in turn relies on cohesion between sister chromatids from S phase until anaphase. S phase cohesion, together with DNA double-strand break (DSB) recruitment of cohesin and formation of damage-induced (DI) cohesion, has previously been shown to be required also for efficient postreplicative DSB repair. The budding yeast acetyltransferase Eco1 (Ctf7) is a common essential factor for S phase and DI-cohesion. The fission yeast Eco1 ortholog, Eso1, is expressed as a fusion protein with the translesion synthesis (TLS) polymerase Polη. The involvement of Eso1 in S phase cohesion was attributed to the Eco1 homologous part of the protein and bypass of UV-induced DNA lesions to the Polη part. Here we describe an additional novel function for budding yeast Polη, i.e. formation of postreplicative DI genome-wide cohesion. This is a unique Polη function not shared with other TLS polymerases. However, Polη deficient cells are DSB repair competent, as Polη is not required for cohesion locally at the DSB. This reveals differential regulation of DSB–proximal cohesion and DI genome-wide cohesion, and challenges the importance of the latter for DSB repair. Intriguingly, we found that specific inactivation of DI genome-wide cohesion increases chromosomal mis-segregation at the entrance of the next cell cycle, suggesting that S phase cohesion is not sufficient for correct chromosome segregation in the presence of DNA damage.
Author Summary
Correct chromosome segregation requires that sister chromatids are held together by the protein complex cohesin, from S phase until anaphase. This S phase established cohesion is, together with DSB recruitment of cohesin and formation of damage-induced (DI) cohesion, also important for repair of DSBs. Eco1 is a common essential factor for S phase and DI-cohesion. The fission yeast Eco1 ortholog, Eso1, is important both for S phase cohesion and for bypass of UV-induced lesions, and is expressed as a fusion protein with Polη. The cohesion function has been attributed solely to Eso1 and the lesion bypass function to the Polη part of the protein. As we found the interaction between the two proteins intriguing, we decided to look for a functional connection also in budding yeast. Indeed, despite being dispensable for S phase cohesion, budding yeast Polη is required for formation of DI genome-wide cohesion. However, Polη-deficient cells are DSB repair competent, revealing differential regulation of DI-cohesion at the break and genome-wide. This finding challenges the importance of DI genome-wide cohesion for DSB repair, and based on our findings we suggest that S phase cohesion is not sufficient for correct chromosome segregation in the presence of DNA damage.
doi:10.1371/journal.pgen.1003158
PMCID: PMC3542068  PMID: 23326240
3.  A New Thermosensitive smc-3 Allele Reveals Involvement of Cohesin in Homologous Recombination in C. elegans 
PLoS ONE  2011;6(9):e24799.
The cohesin complex is required for the cohesion of sister chromatids and for correct segregation during mitosis and meiosis. Crossover recombination, together with cohesion, is essential for the disjunction of homologous chromosomes during the first meiotic division. Cohesin has been implicated in facilitating recombinational repair of DNA lesions via the sister chromatid. Here, we made use of a new temperature-sensitive mutation in the Caenorhabditis elegans SMC-3 protein to study the role of cohesin in the repair of DNA double-strand breaks (DSBs) and hence in meiotic crossing over. We report that attenuation of cohesin was associated with extensive SPO-11–dependent chromosome fragmentation, which is representative of unrepaired DSBs. We also found that attenuated cohesin likely increased the number of DSBs and eliminated the need of MRE-11 and RAD-50 for DSB formation in C. elegans, which suggests a role for the MRN complex in making cohesin-loaded chromatin susceptible to meiotic DSBs. Notably, in spite of largely intact sister chromatid cohesion, backup DSB repair via the sister chromatid was mostly impaired. We also found that weakened cohesins affected mitotic repair of DSBs by homologous recombination, whereas NHEJ repair was not affected. Our data suggest that recombinational DNA repair makes higher demands on cohesins than does chromosome segregation.
doi:10.1371/journal.pone.0024799
PMCID: PMC3177864  PMID: 21957461
4.  Ctf18 is required for homologous recombination-mediated double-strand break repair 
Nucleic Acids Research  2007;35(15):4989-5000.
The efficient repair of double-strand breaks (DSBs) is crucial in maintaining genomic integrity. Sister chromatid cohesion is important for not only faithful chromosome segregation but also for proper DSB repair. During DSB repair, the Smc1–Smc3 cohesin complex is loaded onto chromatin around the DSB to support recombination-mediated DSB repair. In this study, we investigated whether Ctf18, a factor implicated in the establishment of sister chromatid cohesion, is involved in DSB repair in budding yeast. Ctf18 was recruited to HO-endonuclease induced DSB sites in an Mre11-dependent manner and to damaged chromatin in G2/M phase-arrested cells. The ctf18 mutant cells showed high sensitivity to DSB-inducible genotoxic agents and defects in DSB repair, as well as defects in damage-induced recombination between sister chromatids and between homologous chromosomes. These results suggest that Ctf18 is involved in damage-induced homologous recombination.
doi:10.1093/nar/gkm523
PMCID: PMC1976461  PMID: 17636314
5.  A Single Cohesin Complex Performs Mitotic and Meiotic Functions in the Protist Tetrahymena 
PLoS Genetics  2013;9(3):e1003418.
The cohesion of sister chromatids in the interval between chromosome replication and anaphase is important for preventing the precocious separation, and hence nondisjunction, of chromatids. Cohesion is accomplished by a ring-shaped protein complex, cohesin; and its release at anaphase occurs when separase cleaves the complex's α-kleisin subunit. Cohesin has additional roles in facilitating DNA damage repair from the sister chromatid and in regulating gene expression. We tested the universality of the present model of cohesion by studying cohesin in the evolutionarily distant protist Tetrahymena thermophila. Localization of tagged cohesin components Smc1p and Rec8p (the α-kleisin) showed that cohesin is abundant in mitotic and meiotic nuclei. RNAi knockdown experiments demonstrated that cohesin is crucial for normal chromosome segregation and meiotic DSB repair. Unexpectedly, cohesin does not detach from chromosome arms in anaphase, yet chromosome segregation depends on the activity of separase (Esp1p). When Esp1p is depleted by RNAi, chromosomes become polytenic as they undergo multiple rounds of replication, but fail to separate. The cohesion of such bundles of numerous chromatids suggests that chromatids may be connected by factors in addition to topological linkage by cohesin rings. Although cohesin is not detected in transcriptionally active somatic nuclei, its loss causes a slight defect in their amitotic division. Notably, Tetrahymena uses a single version of α-kleisin for both mitosis and meiosis. Therefore, we propose that the differentiation of mitotic and meiotic cohesins found in most other model systems is not due to the need of a specialized meiotic cohesin, but due to additional roles of mitotic cohesin.
Author Summary
During cell division, identical DNA molecules, packaged in the sister chromatids of a chromosome, must be distributed to daughter cells. The cohesion of sister chromatids in the interval between DNA replication and mitotic anaphase is important for preventing the precocious separation, and hence nondisjunction, of chromatids. Cohesion is accomplished by a ring-shaped protein complex, cohesin; and a popular model of cohesion holds that sister chromatids are encircled by cohesin rings and separate upon opening of the rings. During meiosis, cohesin, together with chiasmata, has the additional function of holding bivalents together. Cohesin also has functions in gene regulation and DNA damage repair, and has recently garnered attention as a factor involved in human congenital birth defects. We have studied cohesin in the protist Tetrahymena, which has mitosis/meiosis and transcription performed by different nuclei within the same cell. We exploited this unique feature to experimentally separate the functions of cohesin in chromosome segregation and gene regulation. While the cohesin machinery is generally conserved between eukaryotes, Tetrahymena's phylogenetic distance from standard model organisms allowed us to discover some notable adaptations during the course of evolution.
doi:10.1371/journal.pgen.1003418
PMCID: PMC3610610  PMID: 23555314
6.  Cohesin Protects Genes against γH2AX Induced by DNA Double-Strand Breaks 
PLoS Genetics  2012;8(1):e1002460.
Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high-resolution map of γH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination. We found that recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs in human cells. In addition, we show that cohesin controls γH2AX distribution within domains. Indeed, as we reported previously for transcription, cohesin binding antagonizes γH2AX spreading. Remarkably, depletion of cohesin leads to an increase of γH2AX at cohesin-bound genes, associated with a decrease in their expression level after DSB induction. We propose that, in agreement with their function in chromosome architecture, cohesin could also help to isolate active genes from some chromatin remodelling and modifications such as the ones that occur when a DSB is detected on the genome.
Author Summary
Genomic stability requires that deleterious events such as DNA double-strand breaks (DSBs) are precisely repaired. The natural compaction of DNA into chromatin hinders DNA accessibility and break detection. Therefore, cells respond to DSBs by triggering multiple chromatin modifications that promote accessibility and facilitate repair. We have recently developed a novel system whereby a restriction enzyme can be induced to inflict multiple DSBs across the human genome. This system permits high-resolution characterization of changes in the chromatin landscape that are induced around DSBs. While we previously reported the profile of H2AX phosphorylation (a primary event in chromatin remodelling that takes place in response to DSBs), we now provide the high resolution mapping of cohesin, a complex implicated in the 3-D organisation of chromosomes within the nucleus. Unexpectedly, we have discovered that cohesins play a role in the maintenance of gene transcription in regions where chromatin has been remodelled during the DSB response.
doi:10.1371/journal.pgen.1002460
PMCID: PMC3261922  PMID: 22275873
7.  Sequential Loading of Cohesin Subunits during the First Meiotic Prophase of Grasshoppers 
PLoS Genetics  2007;3(2):e28.
The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, we speculate that more than one cohesin complex participates in the sister chromatid cohesion at prophase I.
Author Summary
Meiosis is a specialized cell division by which sexually reproducing organisms prompt the formation of specialized cells presenting a half of the species chromosomal number. These cells, the so-called gametes, are able to fertilize or be fertilized, depending on the sex in which they are produced and thus restore the species chromosomal number after fertilization. The reduction in the chromosome number is achieved by two successive rounds of chromosome segregations preceded by a single replication of the genetic material. Different proteins, mainly referred to as cohesins, are implied in the correct establishment and maintenance of an intimate association between homologous chromosomes by ensuring their close association until their separation in the first meiotic division. Grasshoppers have been considered as a gorgeous model for meiotic studies for decades due to their low chromosomal number, the large size of their chromosomes, and the well-defined meiotic stages at cytological level. On these grounds, we have combined classical grasshopper chromosome knowledge with protein immunolocalization tools in order to precisely analyze the presence of cohesins throughout the prophase of the first meiotic division. The results not only describe the dynamic loading pattern of several cohesin subunits in two grasshopper species, but they also surprisingly bring into light that different cohesins are sequentially loaded onto meiotic chromosomes throughout the first meiotic prophase. Finally, we discuss the possible roles for this sequential protein loading in relation to the processes that operate during meiosis, proposing a model for meiotic chromosome structure. Besides the novel scientific contributions for a better understanding of the meiotic process, this study clearly points out that classical cytogenetic models can be used to solve modern biological problems.
doi:10.1371/journal.pgen.0030028
PMCID: PMC1802827  PMID: 17319746
8.  Divergent kleisin subunits of cohesin specify mechanisms to tether and release meiotic chromosomes 
eLife  2014;3:e03467.
We show that multiple, functionally specialized cohesin complexes mediate the establishment and two-step release of sister chromatid cohesion that underlies the production of haploid gametes. In C. elegans, the kleisin subunits REC-8 and COH-3/4 differ between meiotic cohesins and endow them with distinctive properties that specify how cohesins load onto chromosomes and then trigger and release cohesion. Unlike REC-8 cohesin, COH-3/4 cohesin becomes cohesive through a replication-independent mechanism initiated by the DNA double-stranded breaks that induce crossover recombination. Thus, break-induced cohesion also tethers replicated meiotic chromosomes. Later, recombination stimulates separase-independent removal of REC-8 and COH-3/4 cohesins from reciprocal chromosomal territories flanking the crossover site. This region-specific removal likely underlies the two-step separation of homologs and sisters. Unexpectedly, COH-3/4 performs cohesion-independent functions in synaptonemal complex assembly. This new model for cohesin function diverges from that established in yeast but likely applies directly to plants and mammals, which utilize similar meiotic kleisins.
DOI: http://dx.doi.org/10.7554/eLife.03467.001
eLife digest
Most plant and animal cells have a pair of each chromosome: one copy is inherited from the father, the other from the mother. When a cell divides, each daughter cell must receive a copy of all of the original cell's genetic information. To this end, the chromosomes are replicated to form so-called ‘sister chromatids’, which are then segregated equally between the two daughter cells.
In contrast, sex cells such as eggs and sperm (also called gametes) have a single copy of each chromosome. When an egg and a sperm fuse to form a single cell (called a zygote), the zygote ends up with a full set of chromosomes. Gametes are formed by two successive rounds of cell division that occur after the chromosomes are replicated. The first round separates the pairs of chromosomes, and the second separates the sister chromatids to produce the gametes, each of which has half the original amount of genetic information.
If something goes awry in the production of gametes, a zygote can end up with the wrong number of chromosomes. Almost one-third of human zygotes inherit an aberrant complement of chromosomes, and many of these zygotes either fail to survive or develop into offspring with birth defects and developmental disorders.
To ensure that gametes receive the correct number of chromosomes, the sister chromatids remain bound together by a ring-shaped protein complex during the first cell division. Previous studies on how this protein complex—called cohesin—tethers the sister chromatids together were conducted on yeast and mammalian cells. Now, Severson and Meyer show that, in a microscopic worm called Caenorhabditis elegans, cohesin functions differently from how it functions in the simpler yeast cells.
Severson and Meyer found that rather than using a single cohesin complex like in yeast, the worms use multiple cohesin complexes that have different versions of one key protein subunit. Changing this single subunit has a major impact on cohesin's function. Consequently, each complex plays a specific role in tethering and then releasing sister chromatids. One of the cohesin complexes is triggered to tether the sister chromatids when the chromosomes replicate. Unexpectedly, another complex only tethers the sisters once breaks occur in the DNA. These breaks allow sister chromatids that are produced from maternally- and paternally-derived chromosomes to cross over and swap genetic material—which increases the genetic diversity of any future offspring. After these genetic swaps occur, the cohesin complexes are then selectively removed by different mechanisms, first to release the pairs of chromosomes and then the sister chromatids.
The findings of Severson and Meyer establish a new model for the mechanisms of chromosome segregation during gamete production. Further studies are now needed to determine the roles and regulation of these protein complexes in other species—including plants and mammals, which use similar cohesin complexes.
DOI: http://dx.doi.org/10.7554/eLife.03467.002
doi:10.7554/eLife.03467
PMCID: PMC4174578  PMID: 25171895
cohesin; sister chromatid cohesion; meiosis; gametogenesis; kleisin; aneuploidy; C. elegans
9.  Alkylation Base Damage Is Converted into Repairable Double-Strand Breaks and Complex Intermediates in G2 Cells Lacking AP Endonuclease 
PLoS Genetics  2011;7(4):e1002059.
DNA double-strand breaks (DSBs) are potent sources of genome instability. While there is considerable genetic and molecular information about the disposition of direct DSBs and breaks that arise during replication, relatively little is known about DSBs derived during processing of single-strand lesions, especially for the case of single-strand breaks (SSBs) with 3′-blocked termini generated in vivo. Using our recently developed assay for detecting end-processing at random DSBs in budding yeast, we show that single-strand lesions produced by the alkylating agent methyl methanesulfonate (MMS) can generate DSBs in G2-arrested cells, i.e., S-phase independent. These derived DSBs were observed in apn1/2 endonuclease mutants and resulted from aborted base excision repair leading to 3′ blocked single-strand breaks following the creation of abasic (AP) sites. DSB formation was reduced by additional mutations that affect processing of AP sites including ntg1, ntg2, and, unexpectedly, ogg1, or by a lack of AP sites due to deletion of the MAG1 glycosylase gene. Similar to direct DSBs, the derived DSBs were subject to MRX (Mre11, Rad50, Xrs2)-determined resection and relied upon the recombinational repair genes RAD51, RAD52, as well as on the MCD1 cohesin gene, for repair. In addition, we identified a novel DNA intermediate, detected as slow-moving chromosomal DNA (SMD) in pulsed field electrophoresis gels shortly after MMS exposure in apn1/2 cells. The SMD requires nicked AP sites, but is independent of resection/recombination processes, suggesting that it is a novel structure generated during processing of 3′-blocked SSBs. Collectively, this study provides new insights into the potential consequences of alkylation base damage in vivo, including creation of novel structures as well as generation and repair of DSBs in nonreplicating cells.
Author Summary
DNA double-strand breaks (DSBs) are an important source of genome instability that can lead to severe biological consequences including tumorigenesis and cell death. Although much is known about DSBs induced directly by ionizing radiation and radiomimetic cancer drugs, there is a relative dearth of information about the formation of derived DSBs that arise from processing of single-strand lesions. Since as many as 10,000–200,000 single-strand lesions have been estimated to occur each day in mammalian cells, conversion of even a small percentage of such lesions to DSBs could dramatically affect genome stability. Here we addressed the mechanism of formation and repair of derived DSBs in vivo during the processing of DNA methylation damage in yeast that are defective in base excision repair (BER) due to a lack of AP endonucleases. Armed with a technique developed in our lab that detects resection at DSBs, a first step in DSB repair, we demonstrated formation of DSBs in G2 cells and the role of recombinational repair in subsequent chromosome restitution. Furthermore, we have identified a novel repair intermediate that can be generated if abasic sites are nicked by AP lyases, providing additional insights into the processing of 3′-blocked groups at single-strand breaks.
doi:10.1371/journal.pgen.1002059
PMCID: PMC3084215  PMID: 21552545
10.  Histone H3K56 Acetylation, Rad52, and Non-DNA Repair Factors Control Double-Strand Break Repair Choice with the Sister Chromatid 
PLoS Genetics  2013;9(1):e1003237.
DNA double-strand breaks (DSBs) are harmful lesions that arise mainly during replication. The choice of the sister chromatid as the preferential repair template is critical for genome integrity, but the mechanisms that guarantee this choice are unknown. Here we identify new genes with a specific role in assuring the sister chromatid as the preferred repair template. Physical analyses of sister chromatid recombination (SCR) in 28 selected mutants that increase Rad52 foci and inter-homolog recombination uncovered 8 new genes required for SCR. These include the SUMO/Ub-SUMO protease Wss1, the stress-response proteins Bud27 and Pdr10, the ADA histone acetyl-transferase complex proteins Ahc1 and Ada2, as well as the Hst3 and Hst4 histone deacetylase and the Rtt109 histone acetyl-transferase genes, whose target is histone H3 Lysine 56 (H3K56). Importantly, we use mutations in H3K56 residue to A, R, and Q to reveal that H3K56 acetylation/deacetylation is critical to promote SCR as the major repair mechanism for replication-born DSBs. The same phenotype is observed for a particular class of rad52 alleles, represented by rad52-C180A, with a DSB repair defect but a spontaneous hyper-recombination phenotype. We propose that specific Rad52 residues, as well as the histone H3 acetylation/deacetylation state of chromatin and other specific factors, play an important role in identifying the sister as the choice template for the repair of replication-born DSBs. Our work demonstrates the existence of specific functions to guarantee SCR as the main repair event for replication-born DSBs that can occur by two pathways, one Rad51-dependent and the other Pol32-dependent. A dysfunction can lead to genome instability as manifested by high levels of homolog recombination and DSB accumulation.
Author Summary
Double-strand breaks (DSBs) are among the most dangerous DNA lesions and can lead to genomic instability, a process associated with cancer and hereditary diseases. An important source of DSBs is replication, Sister Chromatid Recombination (SCR) being the main mechanism for DSB repair in dividing eukaryotic cells. SCR repair is error-free and uses the sister chromatid as template, generating an identical DNA sequence and therefore preventing genomic instability. In this work, we use an inverted-repeat assay with which we can physically detect SCR intermediates generated by the repair of a replication-born DSB. We hypothesized that SCR defects can result in an increase of recombination with the homologous chromosome, so we assayed SCR in 28 mutants previously described to increase homolog recombination. Our results describe 8 new genes involved in SCR, including functions such as histone acetylation/deacetylation, SUMO-Ubiquitin metabolism, and stress response, as well as an allele of RAD52. This demonstrates the importance of the choice of the sister chromatid as template for DSB repair and provides a broad vision of SCR as a tightly regulated process essential for genome integrity.
doi:10.1371/journal.pgen.1003237
PMCID: PMC3554610  PMID: 23357952
11.  Meiosis-Specific Cohesin Component, Stag3 Is Essential for Maintaining Centromere Chromatid Cohesion, and Required for DNA Repair and Synapsis between Homologous Chromosomes 
PLoS Genetics  2014;10(7):e1004413.
Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin complexes. Furthermore, our data suggests that STAG3 is required for structural changes of chromosomes that mediate chromosome pairing and synapsis, DNA repair and progression of meiosis.
Author Summary
Meiosis is a specialized cell division required for the formation of gametes (sperm and egg). Early in meiosis, the chromosome pairs that we inherit from our mother and father become linked and genetic material is exchanged. This is a remarkable process as every gamete that we make is unique, and the unison between a sperm and egg will create a new individual that harbors novel combinations of characteristics from each parents' family tree. Linkage and genetic exchange between chromosomes is facilitated by a linear protein scaffold structure. A group of protein complexes known as cohesins are a key component of the protein scaffold. To date, there are 4 meiosis-specific cohesin complexes identified. Only one cohesin component known as STAG3 is represented in all meiosis-specific cohesins. We mutated the gene that encodes for STAG3 in mouse and discovered that it results in meiotic failure and absence of gametes. From careful analysis we have determined that STAG3 is required for the stability of meiosis-specific cohesins, which ensure that chromosomes are paired and genetic material is exchanged. Our findings imply that abnormalities in human STAG3 will give rise to chromosome defects, infertility and gonad atrophy.
doi:10.1371/journal.pgen.1004413
PMCID: PMC4081007  PMID: 24992337
12.  Distinct Functions of Human Cohesin-SA1 and Cohesin-SA2 in Double-Strand Break Repair 
Molecular and Cellular Biology  2014;34(4):685-698.
Cohesin is an essential multiprotein complex that mediates sister chromatid cohesion critical for proper segregation of chromosomes during cell division. Cohesin is also involved in DNA double-strand break (DSB) repair. In mammalian cells, cohesin is involved in both DSB repair and the damage checkpoint response, although the relationship between these two functions is unclear. Two cohesins differing by one subunit (SA1 or SA2) are present in somatic cells, but their functional specificities with regard to DNA repair remain enigmatic. We found that cohesin-SA2 is the main complex corecruited with the cohesin-loading factor NIPBL to DNA damage sites in an S/G2-phase-specific manner. Replacing the diverged C-terminal region of SA1 with the corresponding region of SA2 confers this activity on SA1. Depletion of SA2 but not SA1 decreased sister chromatid homologous recombination repair and affected repair pathway choice, indicating that DNA repair activity is specifically associated with cohesin recruited to damage sites. In contrast, both cohesin complexes function in the intra-S checkpoint, indicating that cell cycle-specific damage site accumulation is not a prerequisite for cohesin's intra-S checkpoint function. Our findings reveal the unique ways in which cohesin-SA1 and cohesin-SA2 participate in the DNA damage response, coordinately protecting genome integrity in human cells.
doi:10.1128/MCB.01503-13
PMCID: PMC3911484  PMID: 24324008
13.  Frequent and Efficient Use of the Sister Chromatid for DNA Double-Strand Break Repair during Budding Yeast Meiosis 
PLoS Biology  2010;8(10):e1000520.
Studies of DNA double-strand break repair during meiosis reveal that a substantial fraction of recombination occurs between sister chromatids.
Recombination between homologous chromosomes of different parental origin (homologs) is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction–containing recombination intermediates (joint molecules [JMs]) show a strong bias towards inter-homolog and against inter-sister JMs. However, recombination between sister chromatids also has an important role in meiosis. The genomes of diploid organisms in natural populations are highly polymorphic for insertions and deletions, and meiotic double-strand breaks (DSBs) that form within such polymorphic regions must be repaired by inter-sister recombination. Efforts to study inter-sister recombination during meiosis, in particular to determine recombination frequencies and mechanisms, have been constrained by the inability to monitor the products of inter-sister recombination. We present here molecular-level studies of inter-sister recombination during budding yeast meiosis. We examined events initiated by DSBs in regions that lack corresponding sequences on the homolog, and show that these DSBs are efficiently repaired by inter-sister recombination. This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold) yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies. Furthermore, inter-sister JMs formed in mek1Δ mutants are preferentially lost, while inter-homolog JMs are maintained. These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids. We suggest that a Mek1-dependent reduction in the rate of inter-sister repair, combined with the destabilization of inter-sister JMs, promotes inter-homolog recombination while retaining the capacity for inter-sister recombination when inter-homolog recombination is not possible.
Author Summary
In diploid organisms, which contain two parental sets of chromosomes, double-stranded breaks in DNA can be repaired by recombination, either with a copy of the chromosome produced by replication (the sister chromatid), or with either chromatid of the other parental chromosome (the homolog). During meiosis, recombination with the homolog ensures faithful segregation of chromosomes to gametes (sperm or egg). It has been suggested that use of the spatially distant homolog, as opposed to the nearby sister chromatid, results from a meiosis-specific barrier to recombination between sister chromatids. However, there are situations where meiotic recombination must occur between sister chromatids, such as when recombination initiates in sequences that are absent from the homolog. By studying such a situation, we show that meiotic recombination with the sister chromatid occurs with similar timing and efficiency as recombination with the homolog. Further analysis indicates that inter-sister recombination is more common than was previously thought, although still far less prevalent than in somatic cells, where inter-sister recombination predominates. We suggest that meiosis-specific factors act to roughly equalize repair from the sister and homolog, which both allows the establishment of physical connections between homologs and ensures timely repair of breaks incurred in regions lacking corresponding sequences on the homolog.
doi:10.1371/journal.pbio.1000520
PMCID: PMC2957403  PMID: 20976044
14.  Roles of Vertebrate Smc5 in Sister Chromatid Cohesion and Homologous Recombinational Repair ▿ 
Molecular and Cellular Biology  2011;31(7):1369-1381.
The structural maintenance of chromosomes (Smc) family members Smc5 and Smc6 are both essential in budding and fission yeasts. Yeast smc5/6 mutants are hypersensitive to DNA damage, and Smc5/6 is recruited to HO-induced double-strand breaks (DSBs), facilitating intersister chromatid recombinational repair. To determine the role of the vertebrate Smc5/6 complex during the normal cell cycle, we generated an Smc5-deficient chicken DT40 cell line using gene targeting. Surprisingly, Smc5− cells were viable, although they proliferated more slowly than controls and showed mitotic abnormalities. Smc5-deficient cells were sensitive to methyl methanesulfonate and ionizing radiation (IR) and showed increased chromosome aberration levels upon irradiation. Formation and resolution of Rad51 and gamma-H2AX foci after irradiation were altered in Smc5 mutants, suggesting defects in homologous recombinational (HR) repair of DNA damage. Ku70−/− Smc5− cells were more sensitive to IR than either single mutant, with Rad54−/− Smc5− cells being no more sensitive than Rad54−/− cells, consistent with an HR function for the vertebrate Smc5/6 complex. Although gene targeting occurred at wild-type levels, recombinational repair of induced double-strand breaks was reduced in Smc5− cells. Smc5 loss increased sister chromatid exchanges and sister chromatid separation distances in mitotic chromosomes. We conclude that Smc5/6 regulates recombinational repair by ensuring appropriate sister chromatid cohesion.
doi:10.1128/MCB.00786-10
PMCID: PMC3135288  PMID: 21245390
15.  Mouse RAD54 Affects DNA Double-Strand Break Repair and Sister Chromatid Exchange 
Molecular and Cellular Biology  2000;20(9):3147-3156.
Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54 wild-type and knockout cells revealed direct evidence for a role of mRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence of mRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA.
PMCID: PMC85609  PMID: 10757799
16.  The Cohesion Protein SOLO Associates with SMC1 and Is Required for Synapsis, Recombination, Homolog Bias and Cohesion and Pairing of Centromeres in Drosophila Meiosis 
PLoS Genetics  2013;9(7):e1003637.
Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome cores.
Author Summary
Sexual reproduction entails an intricate 2-step division called meiosis in which homologous chromosomes and sister chromatids are sequentially segregated to yield gametes (eggs and sperm) with exactly one copy of each chromosome. The Drosophila meiosis protein SOLO is essential for cohesion between sister chromatids. SOLO localizes to centromeres throughout meiosis where it collaborates with the conserved cohesin complex to enable sister centromeres to orient properly – to the same pole during the first division and to opposite poles during the second division. In solo mutants, sister chromatids become disconnected early in meiosis and segregate randomly through both meiotic divisions generating gametes with random (and mostly wrong) numbers of chromosomes. In this study we show that SOLO also localizes to chromosome arms where it is required to construct stable synaptonemal complexes that connect homologs while they recombine. In addition, SOLO is required to prevent crossovers between sister chromatids, as only homolog crossovers are useful for forming the interhomolog connections (chiasmata) needed for homolog segregation. SOLO collaborates with cohesin for these tasks as well. We propose that SOLO is a subunit of a specialized meiotic cohesin complex and a multi-purpose cohesion protein that regulates several meiotic processes needed for proper chromosome segregation.
doi:10.1371/journal.pgen.1003637
PMCID: PMC3715423  PMID: 23874232
17.  Inhibition of the Smc5/6 Complex during Meiosis Perturbs Joint Molecule Formation and Resolution without Significantly Changing Crossover or Non-crossover Levels 
PLoS Genetics  2013;9(11):e1003898.
Meiosis is a specialized cell division used by diploid organisms to form haploid gametes for sexual reproduction. Central to this reductive division is repair of endogenous DNA double-strand breaks (DSBs) induced by the meiosis-specific enzyme Spo11. These DSBs are repaired in a process called homologous recombination using the sister chromatid or the homologous chromosome as a repair template, with the homolog being the preferred substrate during meiosis. Specific products of inter-homolog recombination, called crossovers, are essential for proper homolog segregation at the first meiotic nuclear division in budding yeast and mice. This study identifies an essential role for the conserved Structural Maintenance of Chromosomes (SMC) 5/6 protein complex during meiotic recombination in budding yeast. Meiosis-specific smc5/6 mutants experience a block in DNA segregation without hindering meiotic progression. Establishment and removal of meiotic sister chromatid cohesin are independent of functional Smc6 protein. smc6 mutants also have normal levels of DSB formation and repair. Eliminating DSBs rescues the segregation block in smc5/6 mutants, suggesting that the complex has a function during meiotic recombination. Accordingly, smc6 mutants accumulate high levels of recombination intermediates in the form of joint molecules. Many of these joint molecules are formed between sister chromatids, which is not normally observed in wild-type cells. The normal formation of crossovers in smc6 mutants supports the notion that mainly inter-sister joint molecule resolution is impaired. In addition, return-to-function studies indicate that the Smc5/6 complex performs its most important functions during joint molecule resolution without influencing crossover formation. These results suggest that the Smc5/6 complex aids primarily in the resolution of joint molecules formed outside of canonical inter-homolog pathways.
Author Summary
Most eukaryotic cells are diploid, which means that they contain two copies of each chromosome – one from each parent. In order to preserve the chromosome number from generation to generation, diploid organisms employ a process called meiosis to form gametes containing only one copy of each chromosome. During sexual reproduction, two gametes (sperm and eggs in mammals) fuse to form a zygote with the same chromosome number as the parents. This zygote will develop into a new organism that has genetic characteristics unique from, but still related to, both parents. The reduction of chromosome number and the reshuffling of genetic traits during meiosis depend on the repair of naturally occurring DNA breaks. Improper break repair during meiosis may block meiosis altogether or form genetically instable gametes, leading to fertility problems or defects in the offspring. The study presented here demonstrates the importance of the evolutionarily conserved Smc5/6 protein complex in upholding the integrity of meiotic repair processes. Our results show that cells deficient in components of the Smc5/6 complex lead to inviable meiotic products. Cells lacking functional Smc5/6 complex are unable to direct DNA repair to the proper template and accumulate abnormal repair intermediates, which inhibit the reductive division.
doi:10.1371/journal.pgen.1003898
PMCID: PMC3820751  PMID: 24244180
18.  The AtRAD21.1 and AtRAD21.3 Arabidopsis cohesins play a synergistic role in somatic DNA double strand break damage repair 
BMC Plant Biology  2014;14(1):353.
Background
The RAD21 cohesin plays, besides its well-recognised role in chromatid cohesion, a role in DNA double strand break (dsb) repair. In Arabidopsis there are three RAD21 paralog genes (AtRAD21.1, AtRAD21.2 and AtRAD21.3), yet only AtRAD21.1 has been shown to be required for DNA dsb damage repair. Further investigation of the role of cohesins in DNA dsb repair was carried out and is here reported.
Results
We show for the first time that not only AtRAD21.1 but also AtRAD21.3 play a role in somatic DNA dsb repair. Comet data shows that the lack of either cohesins induces a similar high basal level of DNA dsb in the nuclei and a slower DNA dsb repair kinetics in both cohesin mutants. The observed AtRAD21.3 transcriptional response to DNA dsb induction reinforces further the role of this cohesin in DNA dsb repair. The importance of AtRAD21.3 in DNA dsb damage repair, after exposure to DNA dsb damage inducing agents, is notorious and recognisably evident at the phenotypical level, particularly when the AtRAD21.1 gene is also disrupted.
Data on the kinetics of DNA dsb damage repair and DNA damage sensitivity assays, of single and double atrad21 mutants, as well as the transcription dynamics of the AtRAD21 cohesins over a period of 48 hours after the induction of DNA dsb damage is also shown.
Conclusions
Our data demonstrates that both Arabidopsis cohesin (AtRAD21.1 and AtRAD21.3) play a role in somatic DNA dsb repair. Furthermore, the phenotypical data from the atrad21.1 atrad21.3 double mutant indicates that these two cohesins function synergistically in DNA dsb repair. The implications of this data are discussed.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0353-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-014-0353-9
PMCID: PMC4273318  PMID: 25511710
Arabidopsis; AtRAD21.1; AtRAD21.3; Cohesins; Comet assay; DNA damage; Gene expression
19.  The Recombinases Rad51 and Dmc1 Play Distinct Roles in DNA Break Repair and Recombination Partner Choice in the Meiosis of Tetrahymena 
PLoS Genetics  2011;7(3):e1001359.
Repair of programmed DNA double-strand breaks (DSBs) by meiotic recombination relies on the generation of flanking 3′ single-stranded DNA overhangs and their interaction with a homologous double-stranded DNA template. In various common model organisms, the ubiquitous strand exchange protein Rad51 and its meiosis-specific homologue Dmc1 have been implicated in the joint promotion of DNA–strand exchange at meiotic recombination sites. However, the division of labor between these two recombinases is still a puzzle. Using RNAi and gene-disruption experiments, we have studied their roles in meiotic recombination and chromosome pairing in the ciliated protist Tetrahymena as an evolutionarily distant meiotic model. Cytological and electrophoresis-based assays for DSBs revealed that, without Rad51p, DSBs were not repaired. However, in the absence of Dmc1p, efficient Rad51p-dependent repair took place, but crossing over was suppressed. Immunostaining and protein tagging demonstrated that only Dmc1p formed strong DSB–dependent foci on meiotic chromatin, whereas the distribution of Rad51p was diffuse within nuclei. This suggests that meiotic nucleoprotein filaments consist primarily of Dmc1p. Moreover, a proximity ligation assay confirmed that little if any Rad51p forms mixed nucleoprotein filaments with Dmc1p. Dmc1p focus formation was independent of the presence of Rad51p. The absence of Dmc1p did not result in compensatory assembly of Rad51p repair foci, and even artificial DNA damage by UV failed to induce Rad51p foci in meiotic nuclei, while it did so in somatic nuclei within one and the same cell. The observed interhomologue repair deficit in dmc1Δ meiosis is consistent with a requirement for Dmc1p in promoting the homologue as the preferred recombination partner. We propose that relatively short and/or transient Rad51p nucleoprotein filaments are sufficient for intrachromosomal recombination, whereas long nucleoprotein filaments consisting primarily of Dmc1p are required for interhomolog recombination.
Author Summary
Sexual reproduction relies on meiosis, the specialized cell division that allows diploid organisms to halve their chromosome content, resulting in the production of gametes containing one copy of each chromosome. In humans, defects in meiosis cause infertility, stillbirths, and congenital diseases. Homologous recombination is a key step in the meiotic program and is essential for maintaining the fidelity of segregation and for creating genetic diversity. Meiotic recombination begins with self-inflicted DNA breaks that are repaired using the homologous chromosome as a template, in a process that depends upon the universal repair protein Rad51 and its meiosis-specific homologue, Dmc1. The relative contributions of Rad51 and Dmc1 to homologous recombination differ among yeasts, plants, and mammals. We have undertaken a study of these proteins in the evolutionarily distant model organism Tetrahymena thermophila with the hope of clarifying the specialization of these recombinases throughout eukaryotic evolution. We show that, while Rad51 is required for DNA repair, only Dmc1 localizes prominently to meiotic DNA break sites. Also, repair via the homologous chromosome depends on Dmc1. These results suggest that nucleoprotein filaments consisting of primarily Dmc1p allow efficient interhomologue repair, while shorter Rad51 filaments may suffice for repair via the sister chromatid.
doi:10.1371/journal.pgen.1001359
PMCID: PMC3069121  PMID: 21483758
20.  Distinct Targets of the Eco1 Acetyltransferase Modulate Cohesion in S Phase and in Response to DNA Damage 
Molecular cell  2009;34(3):311-321.
Summary
Chromosome segregation and the repair of DNA double-strand breaks (DSBs) require cohesin, the protein complex that mediates sister chromatid cohesion. Cohesion requires both a chromatin binding step and a subsequent tethering step called cohesion generation. Here we provide insight into how cohesion generation is restricted to S phase but can be activated in G2/M by a DSB in budding yeast. We show that Wpl1p inhibits cohesion in G2/M. A DSB counteracts Wpl1p and stimulates cohesion generation by first inducing the phosphorylation of the Mcd1p subunit of cohesin. This phosphorylation activates Eco1p-dependent acetylation of Mcd1p, which in turn antagonizes Wpl1p. Previous studies show that Eco1p antagonizes Wpl1p in S phase by acetylating the Smc3p subunit of cohesin. We show that Mcd1p and Smc3p acetylation antagonize Wpl1p only in their proper context. Thus, Eco1p antagonizes Wpl1p in distinct ways to modulate cohesion generation during the cell cycle and after DNA damage.
doi:10.1016/j.molcel.2009.04.008
PMCID: PMC2737744  PMID: 19450529
21.  RAD50 Is Required for Efficient Initiation of Resection and Recombinational Repair at Random, γ-Induced Double-Strand Break Ends 
PLoS Genetics  2009;5(9):e1000656.
Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.
Author Summary
Double-strand breaks (DSBs) in chromosomal DNA are common sources of genomic change that may be beneficial or deleterious to an organism, from yeast to humans. While they can arise through programmed cellular events, DSBs are frequently associated with defective chromosomal replication, and they are induced by various types of DNA damaging agents such as those employed in cancer therapy, especially ionizing radiation. Elaborate systems have evolved for DSB recognition and subsequent repair, either by homologous recombination or by direct joining of ends. Although much is known about repair mechanisms associated with defined, artificially produced DSBs, there is a relative dearth of information about events surrounding random DSBs. Using a novel, yeast-based system that is applicable to other organisms, we have addressed resection at DSBs, considered a first step in repair. We provide the first direct evidence that cells possess a highly efficient system for recognition and initiation of resection at γ-radiation–induced dirty ends and that the resection is largely dependent on the Rad50/Mre11/Xrs2 complex, identified by the RAD50 gene. The system provides unique opportunities to address other components in resection and repair as well as to identify the contribution of random DSBs and resection to genome instability resulting from other DNA damaging agents.
doi:10.1371/journal.pgen.1000656
PMCID: PMC2734177  PMID: 19763170
22.  A Multi-Step Pathway for the Establishment of Sister Chromatid Cohesion  
PLoS Genetics  2007;3(1):e12.
The cohesion of sister chromatids is mediated by cohesin, a protein complex containing members of the structural maintenance of chromosome (Smc) family. How cohesins tether sister chromatids is not yet understood. Here, we mutate SMC1, the gene encoding a cohesin subunit of budding yeast, by random insertion dominant negative mutagenesis to generate alleles that are highly informative for cohesin assembly and function. Cohesins mutated in the Hinge or Loop1 regions of Smc1 bind chromatin by a mechanism similar to wild-type cohesin, but fail to enrich at cohesin-associated regions (CARs) and pericentric regions. Hence, the Hinge and Loop1 regions of Smc1 are essential for the specific chromatin binding of cohesin. This specific binding and a subsequent Ctf7/Eco1-dependent step are both required for the establishment of cohesion. We propose that a cohesin or cohesin oligomer tethers the sister chromatids through two chromatin-binding events that are regulated spatially by CAR binding and temporally by Ctf7 activation, to ensure cohesins crosslink only sister chromatids.
Author Summary
Complexes containing members of the structural maintenance of chromosomes (Smc) family regulate higher order chromosome architecture in diverse aspects of DNA metabolism including chromosome condensation, sister chromatid cohesion, DNA repair, and global control of transcription. Smc complexes are thought to regulate higher order chromosome folding by tethering together two strands of chromatin. However, the mechanism of tethering is poorly understood in part because of a poor understanding of the function of the core Smc subunits. To gain insight into the structure and function of Smc subunits, we developed a novel strategy of mutagenesis called random insertion dominant negative (RID), which generates informative alleles with high efficiency and should provide an effective tool to study any multi-subunit complex. Using RID we generated novel alleles of a Smc subunit from the cohesin complex. The cohesin complex tethers together newly replicated chromosomes (sister chromatids). The analyses of these RID mutants suggest that the tethering activity of cohesin (and possibly other Smc complexes) is generated by two sequential chromatin-binding events (first the capture of one piece of chromatin followed by the capture of the second piece of chromatin), which are regulated both spatially and temporally. We speculate that the spatial and temporal regulation of cohesin ensures that it tethers together only sister chromatids rather than randomly crosslinking the entire genome.
doi:10.1371/journal.pgen.0030012
PMCID: PMC1779304  PMID: 17238288
23.  A multistep genomic screen identifies new genes required for repair of DNA double-strand breaks in Saccharomyces cerevisiae 
BMC Genomics  2013;14:251.
Background
Efficient mechanisms for rejoining of DNA double-strand breaks (DSBs) are vital because misrepair of such lesions leads to mutation, aneuploidy and loss of cell viability. DSB repair is mediated by proteins acting in two major pathways, called homologous recombination and nonhomologous end-joining. Repair efficiency is also modulated by other processes such as sister chromatid cohesion, nucleosome remodeling and DNA damage checkpoints. The total number of genes influencing DSB repair efficiency is unknown.
Results
To identify new yeast genes affecting DSB repair, genes linked to gamma radiation resistance in previous genome-wide surveys were tested for their impact on repair of site-specific DSBs generated by in vivo expression of EcoRI endonuclease. Eight members of the RAD52 group of DNA repair genes (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, MRE11 and XRS2) and 73 additional genes were found to be required for efficient repair of EcoRI-induced DSBs in screens utilizing both MATa and MATα deletion strain libraries. Most mutants were also sensitive to the clastogenic chemicals MMS and bleomycin. Several of the non-RAD52 group genes have previously been linked to DNA repair and over half of the genes affect nuclear processes. Many proteins encoded by the protective genes have previously been shown to associate physically with each other and with known DNA repair proteins in high-throughput proteomics studies. A majority of the proteins (64%) share sequence similarity with human proteins, suggesting that they serve similar functions.
Conclusions
We have used a genetic screening approach to detect new genes required for efficient repair of DSBs in Saccharomyces cerevisiae. The findings have spotlighted new genes that are critical for maintenance of genome integrity and are therefore of greatest concern for their potential impact when the corresponding gene orthologs and homologs are inactivated or polymorphic in human cells.
doi:10.1186/1471-2164-14-251
PMCID: PMC3637596  PMID: 23586741
EcoRI; Homologous recombination; End-joining; Double-strand break; Bleomycin; MMS; Radiation; RAD52; Gene ontology (GO); Overlapping genes
24.  Down-Regulation of Rad51 Activity during Meiosis in Yeast Prevents Competition with Dmc1 for Repair of Double-Strand Breaks 
PLoS Genetics  2014;10(1):e1004005.
Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a preference for repairing double strand breaks (DSBs) using sister chromatids, in contrast to the conserved, meiosis-specific recombinase, Dmc1, which preferentially repairs programmed DSBs using homologs. Despite the different preferences for repair templates, both Rad51 and Dmc1 are required for interhomolog recombination during meiosis. This paradox has recently been explained by the finding that Rad51 protein, but not its strand exchange activity, promotes Dmc1 function in budding yeast. Rad51 activity is inhibited in dmc1Δ mutants, where the failure to repair meiotic DSBs triggers the meiotic recombination checkpoint, resulting in prophase arrest. The question remains whether inhibition of Rad51 activity is important during wild-type meiosis, or whether inactivation of Rad51 occurs only as a result of the absence of DMC1 or checkpoint activation. This work shows that strains in which mechanisms that down-regulate Rad51 activity are removed exhibit reduced numbers of interhomolog crossovers and noncrossovers. A hypomorphic mutant, dmc1-T159A, makes less stable presynaptic filaments but is still able to mediate strand exchange and interact with accessory factors. Combining dmc1-T159A with up-regulated Rad51 activity reduces interhomolog recombination and spore viability, while increasing intersister joint molecule formation. These results support the idea that down-regulation of Rad51 activity is important during meiosis to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs.
Author Summary
Sexual reproduction involves the generation of chromosomally balanced gametes through the specialized cell division of meiosis. A critical component of meiosis is the physical connection of homologous chromosomes through a combination of recombination and sister chromatid cohesion that is necessary for proper chromosome segregation at the first meiotic division. Meiotic recombination is initiated by the introduction of programmed double strand breaks (DSBs) that are processed and bound by RecA-like proteins called recombinases. In vegetative cells, the Rad51 recombinase preferentially mediates strand invasion of sister chromatids, while in meiotic cells, the meiosis-specific Dmc1 recombinase preferentially invades homologs. How Rad51 and Dmc1 activities are coordinated to generate interhomolog recombinants is a key question in meiosis. This work demonstrates that down-regulation of Rad51 activity is important when interhomolog recombination is occurring to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs. Premature activation of Rad51 results in increased intersister recombination and chromosome missegregation, producing inviable gametes. The evolutionary conservation of both Rad51 and Dmc1 suggests that down-regulation of Rad51 during meiosis may be important in metazoans as well as yeast.
doi:10.1371/journal.pgen.1004005
PMCID: PMC3900393  PMID: 24465215
25.  The Cohesin Subunit Rad21 Is Required for Synaptonemal Complex Maintenance, but Not Sister Chromatid Cohesion, during Drosophila Female Meiosis 
PLoS Genetics  2014;10(8):e1004540.
Replicated sister chromatids are held in close association from the time of their synthesis until their separation during the next mitosis. This association is mediated by the ring-shaped cohesin complex that appears to embrace the sister chromatids. Upon proteolytic cleavage of the α-kleisin cohesin subunit at the metaphase-to-anaphase transition by separase, sister chromatids are separated and segregated onto the daughter nuclei. The more complex segregation of chromosomes during meiosis is thought to depend on the replacement of the mitotic α-kleisin cohesin subunit Rad21/Scc1/Mcd1 by the meiotic paralog Rec8. In Drosophila, however, no clear Rec8 homolog has been identified so far. Therefore, we have analyzed the role of the mitotic Drosophila α-kleisin Rad21 during female meiosis. Inactivation of an engineered Rad21 variant by premature, ectopic cleavage during oogenesis results not only in loss of cohesin from meiotic chromatin, but also in precocious disassembly of the synaptonemal complex (SC). We demonstrate that the lateral SC component C(2)M can interact directly with Rad21, potentially explaining why Rad21 is required for SC maintenance. Intriguingly, the experimentally induced premature Rad21 elimination, as well as the expression of a Rad21 variant with destroyed separase consensus cleavage sites, do not interfere with chromosome segregation during meiosis, while successful mitotic divisions are completely prevented. Thus, chromatid cohesion during female meiosis does not depend on Rad21-containing cohesin.
Author Summary
Meiosis is a specialized form of cell division that ensures production of germ cells with the right number of chromosomes, so that at fertilization the embryo receives complete sets of paternal and maternal chromosomes. The accurate distribution of chromosomes during cell divisions is dependent on a ring-shaped protein complex called cohesin. Cohesin is thought to embrace the chromosomes from the time of their duplication during S-phase until their segregation in the ensuing division. This segregation is facilitated by the controlled proteolytic cleavage of one of the cohesin ring components. Most eukaryotes express specialized variants of this protein: for mitosis the variant Rad21/Scc1/Mcd1 and for meiosis the related protein Rec8. Because Drosophila lacks a clear Rec8 homolog, we have analyzed in the present study whether the mitotic variant Rad21 may also function during meiosis. We have destroyed Rad21-based cohesin by premature cleavage of an engineered Rad21 variant during oogenesis. While we find no indication for effects on the accuracy of meiotic chromosome segregation, Rad21 cleavage results in a premature disassembly of the synaptonemal complex (SC), a structure required for meiotic recombination in Drosophila oocytes. Our interaction studies provide intriguing hints how Rad21 might contribute to SC maintenance.
doi:10.1371/journal.pgen.1004540
PMCID: PMC4125089  PMID: 25101996

Results 1-25 (1320751)