Identification and purification of two distinct complexes containing the five RAD51 paralogs.

Cells defective in any of the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are sensitive to DNA cross-linking agents and to ionizing radiation. Because the paralogs are required for the assembly of DNA damage-induced RAD51 foci, and mutant cell lines are defective in homologous recombination and show genomic instability, their defect is thought to be caused by an inability to promote efficient recombinational repair. Here, we show that the five paralogs exist in two distinct complexes in human cells: one contains RAD51B, RAD51C, RAD51D, and XRCC2 (defined as BCDX2), whereas the other consists of RAD51C with XRCC3. Both protein complexes have been purified to homogeneity and their biochemical properties investigated. BCDX2 binds single-stranded DNA and single-stranded gaps in duplex DNA, in accord with the proposal that the paralogs play an early (pre-RAD51) role in recombinational repair. Moreover, BCDX2 complex binds specifically to nicks in duplex DNA. We suggest that the extreme sensitivity of paralog-defective cell lines to cross-linking agents is owing to defects in the processing of incised cross links and the consequential failure to initiate recombinational repair at these sites.

CLIP-50 immunolocalization during mouse spermiogenesis suggests a role in shaping the sperm nucleus.

The spermatid nucleus and cytoplasm undergo dramatic morphological modifications during spermatid differentiation into mature sperm. Some of the external force causing this nuclear shaping is generated by a microtubular structure termed the manchette, which attaches to the perinuclear ring of the spermatid. Here, we report the isolation and characterization of a protein component of this perinuclear ring in an immunological screening of a mouse testis cDNA library. We termed this protein CLIP-50 because of its high similarity at the amino acid level to the C-terminal region of the microtubule-binding protein CLIP-170/restin. CLIP-50 lacks the characteristic microtubule-binding motif, but retains a portion of the predicted coiled-coiled domain and the metal-binding motif. The CLIP-50 transcript and protein are abundant in testis. The protein is also expressed in heart, lung, kidney, and skin. A distinct size variant exists in brain. In the spermatids, CLIP-50 protein localizes specifically to the centriolar region where the sperm tail originates and to the perinuclear ring from which the manchette emerges. CLIP-50 staining is retained in the ring throughout its migration over the surface of the nucleus which accompanies the nuclear shaping into its characteristic sperm configuration. This localization pattern indicates a very specific function for this novel CLIP derivative during mouse spermiogenesis.

The genomic structure of SYCP3, a meiosis-specific gene encoding a protein of the chromosome core.

SYCP3 localizes to the lateral elements of the synaptonemal complex and is essential for male meiosis. The genomic structure of SYCP3 consists of nine exons spanning approximately 14 kb. In mouse and rat, but not in hamster, the putative translation start of SYCP3 is present in the first exon. The putative promoter of SYCP3 was also cloned and shown to drive transcription of a reporter gene in somatic cells.

Checkpoint and DNA-repair proteins are associated with the cores of mammalian meiotic chromosomes.

Meiotic checkpoints are manifested through protein complexes capable of detecting an abnormality in chromosome metabolism and signaling it to effector molecules that subsequently delay or arrest the progression of meiosis. Some checkpoints act during the first meiotic prophase to monitor the repair of chromosomal DSBs, predominantly by meiotic recombination, or to ensure the correct establishment of synapsis and its well-timed dissolution. In mammals, a number of checkpoint and repair proteins localize to the meiotic chromosomal cores, sometimes in the context of the synaptonemal complex (SC). Here we discuss possible functions of these proteins in the accomplishment of meiotic recombination and normal progression of the meiotic pathway. Also, we present arguments for a structural role of cores and SCs in the assembly of the repair and checkpoint protein complexes on the chromosomes.

Expression and nuclear localization of BLM, a chromosome stability protein mutated in Bloom's syndrome, suggest a role in recombination during meiotic prophase.

Bloom's syndrome (BS) is a recessive human genetic disorder characterized by short stature, immunodeficiency and elevated risk of malignancy. BS cells have genomic instability and an increased frequency of sister chromatid exchange. The gene mutated in BS, BLM, encodes a 3'-5' helicase (BLM) with homology to bacterial recombination factor, RecQ. Human males homozygous for BLM mutations are infertile and heterozygous individuals display increased frequencies of structural chromosome abnormalities in their spermatozoa. Also, mutations in the Saccharomyces cerevisiae homolog of BLM, Sgs1, cause a delay in meiotic nuclear division and a reduction in spore viability. These observations suggest that BLM may play a role during meiosis. Our antibodies raised against the C terminus of the human protein specifically recognize both mouse and human BLM in western blots of cell lines and in successive developmental stages of spermatocytes, but fail to detect BLM protein in a cell line with a C-terminally truncated protein. BLM protein expression and location are detected by immunofluorescence and immunoelectron microscopy as discrete foci that are sparsely present on early meiotic prophase chromosome cores, later found abundantly on synapsed cores, frequently in combination with the recombinases RAD51 and DMC1, and eventually as pure BLM foci. The colocalization of RAD51/DMC1 with BLM and the statistically significant excess of BLM signals in the synapsed pseudoautosomal region of the X-Y chromosomes, which is a recombinational hot spot, provide indications that BLM protein may function in the meiotic recombination process.

Werner's syndrome protein (WRN) migrates Holliday junctions and co-localizes with RPA upon replication arrest.

Individuals affected by the autosomal recessive disorder Werner's syndrome (WS) develop many of the symptoms characteristic of premature ageing. Primary fibroblasts cultured from WS patients exhibit karyotypic abnormalities and a reduced replicative life span. The WRN gene encodes a 3'-5' DNA helicase, and is a member of the RecQ family, which also includes the product of the Bloom's syndrome gene (BLM). In this work, we show that WRN promotes the ATP-dependent translocation of Holliday junctions, an activity that is also exhibited by BLM. In cells arrested in S-phase with hydroxyurea, WRN localizes to discrete nuclear foci that coincide with those formed by the single-stranded DNA binding protein replication protein A. These results are consistent with a model in which WRN prevents aberrant recombination events at sites of stalled replication forks by dissociating recombination intermediates.

RAD51 and DMC1 form mixed complexes associated with mouse meiotic chromosome cores and synaptonemal complexes.

The eukaryotic RecA homologues RAD51 and DMC1 function in homology recognition and formation of joint-molecule recombination intermediates during yeast meiosis. The precise immunolocalization of these two proteins on the meiotic chromosomes of plants and animals has been complicated by their high degree of identity at the amino acid level. With antibodies that have been immunodepleted of cross-reactive epitopes, we demonstrate that RAD51 and DMC1 have identical distribution patterns in extracts of mouse spermatocytes in successive prophase I stages, suggesting coordinate functionality. Immunofluorescence and immunoelectron microscopy with these antibodies demonstrate colocalization of the two proteins on the meiotic chromosome cores at early prophase I. We also show that mouse RAD51 and DMC1 establish protein-protein interactions with each other and with the chromosome core component COR1(SCP3) in a two-hybrid system and in vitro binding analyses. These results suggest that the formation of a multiprotein recombination complex associated with the meiotic chromosome cores is essential for the development and fulfillment of the meiotic recombination process.

The association of ATR protein with mouse meiotic chromosome cores.

The ATR (ataxia telangiectasia- and RAD3-related) protein is present on meiotic prophase chromosome cores and paired cores (synaptonemal complexes, SCs). Its striking characteristic is that the protein forms dense aggregates on the cores and SCs of the last chromosomes to pair at the zygotene-pachytene transition. It would appear that the ATR protein either signals delays in pairing or it is directly involved in the completion of the pairing phase. Atm-deficient spermatocytes, which are defective in the chromosome pairing phase, accumulate large amounts of ATR. The behaviour of ATR at meiotic prophase sets it apart from the distribution of the RAD51/DMC1 recombinase complex and our electron microscope observations confirm that they do not co-localize. We failed to detect ATM in association with cores/SCs and we have reported elsewhere that RAD1 protein does not co-localize with DMC1 foci. The expectation that putative DNA-damage checkpoint proteins. ATR, ATM and RAD1, are associated with RAD51/DMC1 recombination sites where DNA breaks are expected to be present, is therefore not supported by our observations.

Meiotic activation of rat pachytene spermatocytes with okadaic acid: the behaviour of synaptonemal complex components SYN1/SCP1 and COR1/SCP3.

The phosphatase inhibitor okadaic acid accelerates meiotic events in rodent germ cells in culture. Isolated pachytene spermatocytes treated with okadaic acid proceed to a metaphase I arrest in a few hours as opposed to the similar process in vivo, which requires several days. Leptotene/zygotene spermatocytes cannot be activated in this way, suggesting that okadaic acid enables cells to bypass a sensor of the meiotic progression, which is pachytene specific. We monitored the chromosome behaviour accompanying the transition to metaphase I in rat spermatocytes with antibodies against COR1/SCP3, a component of the meiotic chromosome cores, and against the synaptic protein, SYN1/SCP1. Okadaic acid induced a rapid synaptonemal complex dissolution and bivalent separation, followed by chromosome condensation and chiasmata formation, similar to the succession of events in untreated cells. The similarity between meiosis I induced with okadaic acid and the meiosis I events in vivo extends to the dissolution of the nuclear membrane and the disappearance of the microtubule network at the onset of metaphase I. This cell culture system provides a model for the in vivo transition from pachytene to metaphase I and therefore can be used in the study of this transition at the molecular level. The effect of okadaic acid is most likely mediated by the activation of tyrosine kinases, as addition of genistein, a general tyrosine kinase inhibitor, completely abolishes the observed effect of okadaic acid on chromosome metabolism. The okadaic acid-induced progression to the metaphase I arrest is not affected by the inhibition of protein synthesis. However, pachytene spermatocytes incubated in the presence of protein synthesis inhibitors for 6 hours show loss of synapsis which is abnormal in that it is not accompanied by chiasmata formation. The two meiosis-specific proteins, SYN1/SCP1 and COR1/SCP3, are efficiently phosphorylated in vitro by extracts from isolated pachytene cells. Extracts from cells that have reached metaphase I upon okadaic acid treatment, with concomitant displacement of SYN1/SCP1 and COR1/SCP3 from their chromosomes, do not have this capability. These data support the hypothesis that phosphorylation of SYN1/SCP1 and COR1/SCP3 targets their removal from the chromosomes and that activity of the kinases involved correlates with the presence of these two proteins on the chromosomes.

Identification of the mouse beta'-COP Golgi component as a spermatocyte autoantigen in scleroderma and mapping of its gene Copb2 to mouse chromosome 9.

In an immunological screening of a mouse testicular cDNA library with a human CREST serum we isolated five overlapping cDNA clones encoding the mouse homolog of a Golgi coatomer complex protein (accession number AF043120), designated beta'-COP in bovine and p102 in humans. We generated antibodies against this protein which specifically recognize the Golgi apparatus of mouse spermatocytes. FISH analyses assigned the beta'-COP gene Copb2 to mouse Chromosome 9, region E3-F1. Our results demonstrate that CREST sera can contain antibody components against Golgi proteins as well as against nuclear proteins.

Atm deficiency results in severe meiotic disruption as early as leptonema of prophase I.

Infertility is a common feature of the human disorder ataxia-telangiectasia and Atm-deficient mice are completely infertile. To gain further insight into the role of ATM in meiosis, we examined meiotic cells in Atm-deficient mice during development. Spermatocyte degeneration begins between postnatal days 8 and 16.5, soon after entry into prophase I of meiosis, while oocytes degenerate late in embryogenesis prior to dictyate arrest. Using electron microscopy and immunolocalization of meiotic proteins in mutant adult spermatocytes, we found that male and female gametogenesis is severely disrupted in Atm-deficient mice as early as leptonema of prophase I, resulting in apoptotic degeneration. A small number of mutant cells progress into later stages of meiosis, but no cells proceed beyond prophase I. ATR, a protein related to ATM, DMC1, a RAD51 family member, and RAD51 are mislocalized to chromatin and have reduced localization to developing synaptonemal complexes in spermatocytes from Atm-deficient mice, suggesting dysregulation of the orderly progression of meiotic events. ATM protein is normally present at high levels primarily in ova cytoplasm of developing ovarian follicles, and in the nucleus of spermatogonia and to a lesser extent in spermatoctyes, but without localization to the synaptonemal complex. We propose a model in which ATM acts to monitor meiosis by participation in the regulation or surveillance of meiotic progression, similar to its role as a monitor of mitotic cell cycle progression.

Human and mouse homologs of Schizosaccharomyces pombe rad1(+) and Saccharomyces cerevisiae RAD17: linkage to checkpoint control and mammalian meiosis.

Preventing or delaying progress through the cell cycle in response to DNA damage is crucial for eukaryotic cells to allow the damage to be repaired and not incorporated irrevocably into daughter cells. Several genes involved in this process have been discovered in fission and budding yeast. Here, we report the identification of human and mouse homologs of the Schizosaccharomyces pombe DNA damage checkpoint control gene rad1(+) and its Saccharomyces cerevisiae homolog RAD17. The human gene HRAD1 is located on chromosome 5p13 and is most homologous to S. pombe rad1(+). This gene encodes a 382-amino-acid residue protein that is localized mainly in the nucleus and is expressed at high levels in proliferative tissues. This human gene significantly complements the sensitivity to UV light of a S. pombe strain mutated in rad1(+). Moreover, HRAD1 complements the checkpoint control defect of this strain after UV exposure. In addition to functioning in DNA repair checkpoints, S. cerevisiae RAD17 plays a role during meiosis to prevent progress through prophase I when recombination is interrupted. Consistent with a similar role in mammals, Rad1 protein is abundant in testis, and is associated with both synapsed and unsynapsed chromosomes during meiotic prophase I of spermatogenesis, with a staining pattern distinct from that of the recombination proteins Rad51 and Dmc1. Together, these data imply an important role for hRad1 both in the mitotic DNA damage checkpoint and in meiotic checkpoint mechanisms, and suggest that these events are highly conserved from yeast to humans.

Protein-protein interactions in the synaptonemal complex.

In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II.

Rad51 immunocytology in rat and mouse spermatocytes and oocytes.

On the assumption that Rad51 protein plays a role in early meiotic chromosomal events, we examine the location and time of appearance of immuno-reactive Rad51 protein in meiotic prophase chromosomes. The Rad51 foci in mouse spermatocytes appear after the emergence of, and attached to, short chromosomal core segments that we visualize with Cor1-specific antibody. These foci increase in number to about 250 per nucleus at the time when core formation is extensive. The numbers are higher in mouse oocytes and lower in rat spermatocytes, possibly correlating with recombination rates in those cases. In the male mouse, foci decrease in number to approximately 100 while chromosome synapsis is in progress. When synapsis is completed, the numbers of autosomal foci decline to near 0 while the X chromosome retains about 15 foci throughout this time. This stage coincides with the appearance of testis-specific histone H1t at mid- to late pachytene. Electron microscopy reveals that at first Rad51 immunogold-labeled 100 nm nodules are associated with single cores, and that they come to lie between the chromosome cores during synapsis. It appears that these nodules may be the homologs of the Rad51-positive early nodules that are well documented in plants. The reciprocal recombination-correlated late nodules appear after the Rad51 foci are no longer detectable. The absence of Rad51 foci in the chromatin loops suggests that in wild-type mice Rad51/DNA filaments are restricted to DNA at the cores/synaptonemal complexes. The expected association of Rad51 protein with Rad52 could not be verified immunocytologically.