WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks.

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.

Poly(ADP-ribosyl)ation links the chromatin remodeler SMARCA5/SNF2H to RNF168-dependent DNA damage signaling.

Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) arising in native chromatin elicit an RNF8/RNF168-dependent ubiquitylation response, which triggers the recruitment of various repair factors. Precisely how this response is regulated in the context of chromatin remains largely unexplored. Here, we show that SMARCA5/SNF2H, the catalytic subunit of ISWI chromatin remodeling complexes, is recruited to DSBs in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner. Remarkably, PARP activity, although dispensable for the efficient spreading of γH2AX into damaged chromatin, selectively promotes spreading of SMARCA5, the E3 ubiquitin ligase RNF168, ubiquitin conjugates and the ubiquitin-binding factors RAD18 and the RAP80-BRCA1 complex throughout DSB-flanking chromatin. This suggests that PARP regulates the spatial organization of the RNF168-driven ubiquitin response to DNA damage. In support of this, we show that SMARCA5 and RNF168 interact in a DNA damage- and PARP-dependent manner. RNF168 became poly(ADP-ribosyl)ated after DNA damage, while RNF168 and poly(ADP-ribose) chains were required for SMARCA5 binding in vivo, explaining how SMARCA5 is linked to the RNF168 ubiquitin cascade. Moreover, SMARCA5 was found to regulate the ubiquitin response by promoting RNF168 accumulation at DSBs, which subsequently facilitates efficient ubiquitin conjugation and BRCA1 assembly. Underlining the importance of these findings, we show that SMARCA5 depletion renders cells sensitive to IR and results in DSB repair defects. Our study unveils a functional link between DNA damage-induced poly(ADP-ribosyl)ation, SMARCA5-mediated chromatin remodeling and RNF168-dependent signaling and repair of DSBs.

Rad51C is essential for embryonic development and haploinsufficiency causes increased DNA damage sensitivity and genomic instability.

Homologous recombination is essential for repair of DNA interstrand cross-links and double-strand breaks. The Rad51C protein is one of the five Rad51 paralogs in vertebrates implicated in homologous recombination. A previously described hamster cell mutant defective in Rad51C (CL-V4B) showed increased sensitivity to DNA damaging agents and displayed genomic instability. Here, we identified a splice donor mutation at position +5 of intron 5 of the Rad51C gene in this mutant, and generated mice harboring an analogous base pair alteration. Rad51C(splice) heterozygous animals are viable and do not display any phenotypic abnormalities, however homozygous Rad51C(splice) embryos die during early development (E8.5). Detailed analysis of two CL-V4B revertants, V4B-MR1 and V4B-MR2, that have reduced levels of full-length Rad51C transcript when compared to wild type hamster cells, showed increased sensitivity to mitomycin C (MMC) in clonogenic survival, suggesting haploinsufficiency of Rad51C. Similarly, mouse Rad51C(splice/neo) heterozygous ES cells also displayed increased MMC sensitivity. Moreover, in both hamster revertants, Rad51C haploinsufficiency gives rise to increased frequencies of spontaneous and MMC-induced chromosomal aberrations, impaired sister chromatid cohesion and reduced cloning efficiency. These results imply that adequate expression of Rad51C in mammalian cells is essential for maintaining genomic stability and sister chromatid cohesion to prevent malignant transformation.

Identification of conserved pathways of DNA-damage response and radiation protection by genome-wide RNAi.

Ionizing radiation is extremely harmful for human cells, and DNA double-strand breaks (DSBs) are considered to be the main cytotoxic lesions induced. Improper processing of DSBs contributes to tumorigenesis, and mutations in DSB response genes underlie several inherited disorders characterized by cancer predisposition. Here, we performed a comprehensive screen for genes that protect animal cells against ionizing radiation. A total of 45 C. elegans genes were identified in a genome-wide RNA interference screen for increased sensitivity to ionizing radiation in germ cells. These genes include orthologs of well-known human cancer predisposition genes as well as novel genes, including human disease genes not previously linked to defective DNA-damage responses. Knockdown of eleven genes also impaired radiation-induced cell-cycle arrest, and seven genes were essential for apoptosis upon exposure to irradiation. The gene set was further clustered on the basis of increased sensitivity to DNA-damaging cancer drugs cisplatin and camptothecin. Almost all genes are conserved across animal phylogeny, and their relevance for humans was directly demonstrated by showing that their knockdown in human cells results in radiation sensitivity, indicating that this set of genes is important for future cancer profiling and drug development.

Genetic steps of mammalian homologous repair with distinct mutagenic consequences.

Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52(-)(/)(-) mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.

Schizosaccharomyces pombe Rad22A and Rad22B have similar biochemical properties and form multimeric structures.

The Saccharomyces cerevisiae Rad52 protein has a crucial role in the repair of DNA double-strand breaks by homologous recombination. In vitro, Rad52 displays DNA binding and strand annealing activities and promotes Rad51-mediated strand exchange. Schizosaccharomyces pombe has two Rad52 homologues, Rad22A and Rad22B. Whereas rad22A deficient strains exhibit severe defects in repair and recombination, rad22B mutants have a much less severe phenotype. To better understand the role of Rad22A and Rad22B in double-strand break repair, both proteins were purified to near homogeneity. Using gel retardation and filter binding assays, binding of Rad22A and Rad22B to short single-stranded DNAs was demonstrated. Binding of Rad22A to double-stranded oligonucleotides or linearized plasmid molecules containing blunt ends or short single-stranded overhangs could not be detected. Rad22B also does not bind efficiently to short duplex oligonucleotides but binds readily to DNA fragments containing 3'-overhangs. Rad22A as well as Rad22B efficiently promote annealing of complementary single-stranded DNAs. In the presence of Rad22A annealing of complementary DNAs is almost 90%. Whereas in reactions containing Rad22B the maximum level of annealing is 60%, most likely due to inhibition of the reaction by duplex DNA. Gel-filtration experiments and electron microscopic analyses indicate self-association of Rad22A and Rad22B and the formation of multimeric structures as has been observed for Rad52 in yeast and man.

Inactivation of RAD52 aggravates RAD54 defects in mice but not in Schizosaccharomyces pombe.

RAD52 and RAD54 genes from Saccharomyces cerevisiae are required for double-strand break repair through homologous recombination and show epistatic interactions i.e., single and double mutant strains are equally sensitive to DNA damaging agents. In here we combined mutations in RAD52 and RAD54 homologs in Schizosaccharomyces pombe and mice. The analysis of mutant strains in S. pombe demonstrated nearly identical sensitivities of rhp54, rad22A and rad22B double and triple mutants to X-rays, cis-diamminedichloroplatinum and hydroxyurea. In this respect, the fission yeast homologs of RAD54 and RAD52 closely resemble their counterparts in S. cerevisiae. To verify if inactivation of RAD52 affects the DNA damage sensitivities of RAD54 deficient mice, several endpoints were studied in double mutant mice and in bone marrow cells derived from these animals. Haemopoietic depression in bone marrow and the formation of micronuclei after in vivo exposure to mitomycine C (MMC) was not increased in either single or double mutant mice in comparison to wildtype animals. The induction of sister chromatid exchanges in splenocytes was slightly reduced in the RAD54 mutant. A similar reduction was detected in the double mutant. However, a deficiency of RAD52 exacerbates the MMC survival of RAD54 mutant mice and also has a distinct effect on the survival of bone marrow cells after exposure to ionizing radiation. These findings may be explained by additive defects in HR in the double mutant but may also indicate a more prominent role for single-strand annealing in the absence of Rad54.

Lig4 and rad54 are required for repair of DNA double-strand breaks induced by P-element excision in Drosophila.

Site-specific double-strand breaks (DSBs) were generated in the white gene located on the X chromosome of Drosophila by excision of the w(hd) P-element. To investigate the role of nonhomologous end joining (NHEJ) and homologous recombination (HR) in the repair of these breaks, the w(hd) P-element was mobilized in flies carrying mutant alleles of either lig4 or rad54. The survival of both lig4- and rad54-deficient males was reduced to 25% in comparison to the wild type, indicating that both NHEJ and HR are involved in the repair P-induced gaps in males. Survival of lig4-deficient females was not affected at all, implying that HR using the homologous chromosome as a template can partially compensate for the impaired NHEJ pathway. In rad54 mutant females survival was reduced to 70% after w(hd) excision. PCR analysis indicated that the undamaged homologous chromosome may compensate for the potential loss of the broken chromosome in rad54 mutant females after excision. Molecular analysis of the repair junctions revealed microhomology (2-8 bp)-dependent DSB repair in most products. In the absence of Lig4, the 8-bp target site duplication is used more frequently for repair. Our data indicate the presence of efficient alternative end-joining mechanisms, which partly depend on the presence of microhomology but do not require Lig4.

Differential expression and requirements for Schizosaccharomyces pombe RAD52 homologs in DNA repair and recombination.

In fission yeast two RAD52 homologs have been identified, rad22A(+) and rad22B(+). Two-hybrid experiments and GST pull-down assays revealed physical interaction between Rad22A and Rad22B, which is dependent on the N-terminal regions. Interaction with Rhp51 is dependent on the C-terminal parts of either protein. Both Rad22A and Rad22B also interact with RPA. The expression of rad22B(+) in mitotically dividing cells is very low in comparison with rad22A(+) but is strongly enhanced after induction of meiosis, in contrast to rad22A(+). Rad22B mutant cells are not hypersensitive to DNA-damaging agents (X-rays, UV and cisplatin) and display normal levels of recombination. In these respects the Schizosaccharomyces pombe rad22B mutant resembles the weak phenotype of vertebrate cells deficient for RAD52. Mutation of rad22A(+) leads to severe sensitivity to DNA-damaging agents and to defects in recombination. In a rad22Arad22B double mutant a further increase in sensitivity to DNA-damaging agents and additional mitotic recombination defects were observed. The data presented here indicate that Rad22A and Rad22B have overlapping roles in repair and recombination, although specialized functions for each protein cannot be excluded.

Disruption of Drosophila Rad50 causes pupal lethality, the accumulation of DNA double-strand breaks and the induction of apoptosis in third instar larvae.

The Rad50/Mre11/Nbs1 protein complex has a crucial role in DNA metabolism, in particular in double-strand break (DSB) repair through homologous recombination (HR). To elucidate the role of the Rad50 protein complex in DSB repair in a multicellular eukaryote, we generated a Rad50 deficient Drosophila strain by P-element mediated mutagenesis. Disruption of Rad50 causes retarded development and pupal lethality. To investigate the mechanism of pupal death, brains and wing imaginal discs from third instar larvae were studied in more detail. Wing imaginal discs from Rad50 mutant larvae displayed a 3.5-fold increase in the induction of spontaneous apoptotic cells in comparison to their heterozygous siblings. This finding correlates with increased levels of phosphorylated histone H2Av, indicating an accumulation of DSBs in Rad50 mutant larvae. A 45-fold increase in the frequency of anaphase bridges was detected in the brains of Rad50 deficient larvae, consistent with a role for Rad50 in telomere maintenance and/or replication of DNA. The induction of DSBs and defects in chromosome segregation are in agreement with a role of Drosophila Rad50 in repairing the DSBs that arise during replication.

The Drosophila melanogaster DNA Ligase IV gene plays a crucial role in the repair of radiation-induced DNA double-strand breaks and acts synergistically with Rad54.

DNA Ligase IV has a crucial role in double-strand break (DSB) repair through nonhomologous end joining (NHEJ). Most notably, its inactivation leads to embryonic lethality in mammals. To elucidate the role of DNA Ligase IV (Lig4) in DSB repair in a multicellular lower eukaryote, we generated viable Lig4-deficient Drosophila strains by P-element-mediated mutagenesis. Embryos and larvae of mutant lines are hypersensitive to ionizing radiation but hardly so to methyl methanesulfonate (MMS) or the crosslinking agent cis-diamminedichloroplatinum (cisDDP). To determine the relative contribution of NHEJ and homologous recombination (HR) in Drosophila, Lig4; Rad54 double-mutant flies were generated. Survival studies demonstrated that both HR and NHEJ have a major role in DSB repair. The synergistic increase in sensitivity seen in the double mutant, in comparison with both single mutants, indicates that both pathways partially overlap. However, during the very first hours after fertilization NHEJ has a minor role in DSB repair after exposure to ionizing radiation. Throughout the first stages of embryogenesis of the fly, HR is the predominant pathway in DSB repair. At late stages of development NHEJ also becomes less important. The residual survival of double mutants after irradiation strongly suggests the existence of a third pathway for the repair of DSBs in Drosophila.

Ionizing radiation-induced foci formation of mammalian Rad51 and Rad54 depends on the Rad51 paralogs, but not on Rad52.

Homologous recombination is of major importance for the prevention of genomic instability during chromosome duplication and repair of DNA damage, especially double-strand breaks. Biochemical experiments have revealed that during the process of homologous recombination the RAD52 group proteins, including Rad51, Rad52 and Rad54, are involved in an essential step: formation of a joint molecule between the broken DNA and the intact repair template. Accessory proteins for this reaction include the Rad51 paralogs and BRCA2. The significance of homologous recombination for the cell is underscored by the evolutionary conservation of the Rad51, Rad52 and Rad54 proteins from yeast to humans. Upon treatment of cells with ionizing radiation, the RAD52 group proteins accumulate at the sites of DNA damage into so-called foci. For the yeast Saccharomyces cerevisiae, foci formation of Rad51 and Rad54 is abrogated in the absence of Rad52, while Rad51 foci formation does occur in the absence of the Rad51 paralog Rad55. By contrast, we show here that in mammalian cells, Rad52 is not required for foci formation of Rad51 and Rad54. Furthermore, radiation-induced foci formation of Rad51 and Rad54 is impaired in all Rad51 paralog and BRCA2 mutant cell lines tested, while Rad52 foci formation is not influenced by a mutation in any of these recombination proteins. Despite their evolutionary conservation and biochemical similarities, S. cerevisiae and mammalian Rad52 appear to differentially contribute to the DNA-damage response.

Induction of epithelial tumors in Drosophila melanogaster heterozygous for the tumor suppressor gene wts.

The imaginal disk cells of Drosophila have a cell cycle that is very similar to that of mammalian cells. Data concerning factors inducing tumors in these cells may directly relate to the risk of these factors for inducing cancer in humans. One of the genes involved in the regulation of cell cycle control is wts (warts), the Drosophila homolog of the mammalian tumor suppressor gene LATS1. The Drosophila wts mutations are recessive lethal. However, homozygous clones that arise in heterozygous flies in the imaginal disk cells lead to epithelial tumors, spectacular outgrowths visible on the cuticle of the adult. We have treated Drosophila larvae, heterozygous for wts, with the chemical mutagen MMS (methyl methanesulfonate) or with X-rays and measured the appearance of epithelial tumors in the eclosing adult flies. This test is a variation of the well-known Drosophila somatic mutation and recombination test (SMART), where mostly recessive markers have been used leading to visible phenotypes in the eyes and wings of the fly. We show that the sensitivity of this test is far greater than the comparable test system using the recessive eye marker white.

Meiosis in mice without a synaptonemal complex.

The synaptonemal complex (SC) promotes fusion of the homologous chromosomes (synapsis) and crossover recombination events during meiosis. The SC displays an extensive structural conservation between species; however, a few organisms lack SC and execute meiotic process in a SC-independent manner. To clarify the SC function in mammals, we have generated a mutant mouse strain (Sycp1(-/-)Sycp3(-/-), here called SC-null) in which all known SC proteins have been displaced from meiotic chromosomes. While transmission electron microscopy failed to identify any remnants of the SC in SC-null spermatocytes, neither formation of the cohesion axes nor attachment of the chromosomes to the nuclear membrane was perturbed. Furthermore, the meiotic chromosomes in SC-null meiocytes achieved pre-synaptic pairing, underwent early homologous recombination events and sustained a residual crossover formation. In contrast, in SC-null meiocytes synapsis and MLH1-MLH3-dependent crossovers maturation were abolished, whereas the structural integrity of chromosomes was drastically impaired. The variable consequences that SC inactivation has on the meiotic process in different organisms, together with the absence of SC in some unrelated species, imply that the SC could have originated independently in different taxonomic groups.

The NuRD chromatin-remodeling complex regulates signaling and repair of DNA damage.

Cells respond to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) by orchestrating events that coordinate cell cycle progression and DNA repair. How cells signal and repair DSBs is not yet fully understood. A genome-wide RNA interference screen in Caenorhabditis elegans identified egr-1 as a factor that protects worm cells against IR. The human homologue of egr-1, MTA2 (metastasis-associated protein 2), is a subunit of the nucleosome-remodeling and histone deacetylation (NuRD) chromatin-remodeling complex. We show that knockdown of MTA2 and CHD4 (chromodomain helicase DNA-binding protein 4), the catalytic subunit (adenosine triphosphatase [ATPase]) of NuRD, leads to accumulation of spontaneous DNA damage and increased IR sensitivity. MTA2 and CHD4 accumulate in DSB-containing chromatin tracks generated by laser microirradiation. Directly at DSBs, CHD4 stimulates RNF8/RNF168-dependent formation of ubiquitin conjugates to facilitate the accrual of RNF168 and BRCA1. Finally, we show that CHD4 promotes DSB repair and checkpoint activation in response to IR. Thus, the NuRD chromatin-remodeling complex is a novel regulator of DNA damage responses that orchestrates proper signaling and repair of DSBs.

A novel radiosensitive SCID patient with a pronounced G(2)/M sensitivity.

V(D)J rearrangement in lymphoid cells involves repair of double-strand breaks (DSBs) through non-homologous end joining (NHEJ). Defects in this process lead to increased radiosensitivity and severe combined immunodeficiency (RS-SCID). Here, a SCID patient, M3, is described with a T(-)B(+)NK(+) phenotype but without causative mutations in CD3delta, epsilon, zeta or IL7Ralpha, genes specifically involved in T cell development. Clonogenic survival of M3 fibroblasts showed an increased sensitivity to the DSB-inducing agents ionizing radiation and bleomycin, as well as the crosslinking compound, mitomycin C. We did not observe inactivating mutations in known NHEJ genes and results of various DSB-repair assays in G(1) M3 cells were indistinguishable from those obtained with normal cells. However, we found increased chromosomal radiosensitivity at the G(2) phase of the cell cycle. Checkpoint analysis indicated functional G(1)/S and intra-S checkpoints after irradiation but impaired activation of the "early" G(2)/M checkpoint. Together these results indicate a novel class of RS-SCID patients characterized by the specific absence of T lymphocytes and associated with defects in G(2)-specific DSB repair. The pronounced G(2)/M radiosensitivity of the RS-SCID patient described here, suggests a defect in a putative novel and uncharacterized factor involved in cellular DNA damage responses and T cell development.

Increased DNA damage sensitivity of Cornelia de Lange syndrome cells: evidence for impaired recombinational repair.

Cornelia de Lange syndrome (CdLS) is a rare dominantly inherited multisystem disorder affecting both physical and mental development. Heterozygous mutations in the NIPBL gene were found in about half of CdLS cases. Scc2, the fungal ortholog of the NIPBL gene product, is essential for establishing sister chromatid cohesion. In yeast, the absence of cohesion leads to chromosome mis-segregation and defective repair of DNA double-strand breaks. To evaluate possible DNA repair defects in CdLS cells, we characterized the cellular responses to DNA-damaging agents. We show that cells derived from CdLS patients, both with and without detectable NIPBL mutations, have an increased sensitivity for mitomycin C (MMC). Exposure of CdLS fibroblast and B-lymphoblastoid cells to MMC leads to enhanced cell killing and reduced proliferation and, in the case of primary fibroblasts, an increased number of chromosomal aberrations. After X-ray exposure increased numbers of chromosomal aberrations were also detected, but only in cells irradiated in the G(2)-phase of the cell cycle when repair of double-strand breaks is dependent on the establishment of sister chromatid cohesion. Repair at the G(1) stage is not affected in CdLS cells. Our studies indicate that CdLS cells have a reduced capacity to tolerate DNA damage, presumably as a result of reduced DNA repair through homologous recombination.

Two levels of interference in mouse meiotic recombination.

During meiosis, homologous chromosomes (homologs) undergo recombinational interactions, which can yield crossovers (COs) or noncrossovers. COs exhibit interference; they are more evenly spaced along the chromosomes than would be expected if they were placed randomly. The protein complexes involved in recombination can be visualized as immunofluorescent foci. We have analyzed the distribution of such foci along meiotic prophase chromosomes of the mouse to find out when interference is imposed and whether interference manifests itself at a constant level during meiosis. We observed strong interference among MLH1 foci, which mark CO positions in pachytene. Additionally, we detected substantial interference well before this point, in late zygotene, among MSH4 foci, and similarly, among replication protein A (RPA) foci. MSH4 foci and RPA foci both mark interhomolog recombinational interactions, most of which do not yield COs in the mouse. Furthermore, this zygotene interference did not depend on SYCP1, which is a transverse filament protein of mouse synaptonemal complexes. Interference is thus not specific to COs but may occur in other situations in which the spatial distribution of events has to be controlled. Differences between the distributions of MSH4/RPA foci and MLH1 foci along synaptonemal complexes might suggest that CO interference occurs in two successive steps.

Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombination, and XY body formation.

In meiotic prophase, synaptonemal complexes (SCs) closely appose homologous chromosomes (homologs) along their length. SCs are assembled from two axial elements (AEs), one along each homolog, which are connected by numerous transverse filaments (TFs). We disrupted the mouse gene encoding TF protein Sycp1 to analyze the role of TFs in meiotic chromosome behavior and recombination. Sycp1(-/-) mice are infertile, but otherwise healthy. Sycp1(-/-) spermatocytes form normal AEs, which align homologously, but do not synapse. Most Sycp1(-/-) spermatocytes arrest in pachynema, whereas a small proportion reaches diplonema, or, exceptionally, metaphase I. In leptotene Sycp1(-/-) spermatocytes, gammaH2AX (indicative of DNA damage, including double-strand breaks) appears normal. In pachynema, Sycp1(-/-) spermatocytes display a number of discrete gammaH2AX domains along each chromosome, whereas gammaH2AX disappears from autosomes in wild-type spermatocytes. RAD51/DMC1, RPA, and MSH4 foci (which mark early and intermediate steps in pairing/recombination) appear in similar numbers as in wild type, but do not all disappear, and MLH1 and MLH3 foci (which mark late steps in crossing over) are not formed. Crossovers were rare in metaphase I of Sycp1(-/-) mice. We propose that SYCP1 has a coordinating role, and ensures formation of crossovers. Unexpectedly, Sycp1(-/-) spermatocytes did not form XY bodies.

DNA double-strand break repair by homologous recombination.

The induction of double-strand breaks (DSBs) in DNA by exposure to DNA damaging agents, or as intermediates in normal cellular processes, constitutes a severe threat for the integrity of the genome. If not properly repaired, DSBs may result in chromosomal aberrations, which, in turn, can lead to cell death or to uncontrolled cell growth. To maintain the integrity of the genome, multiple pathways for the repair of DSBs have evolved during evolution: homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA). HR has the potential to lead to accurate repair of DSBs, whereas NHEJ and SSA are essentially mutagenic. In yeast, DSBs are primarily repaired via high-fidelity repair of DSBs mediated by HR, whereas in higher eukaryotes, both HR and NHEJ are important. In this review, we focus on the functional conservation of HR from fungi to mammals and on the role of the individual proteins in this process.