MEIOB Targets Single-Strand DNA and Is Necessary for Meiotic Recombination

Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB). This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 ^−/− spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob ^−/− meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.     

The dual role of HOP2 in mammalian meiotic homologous recombination

Deletion of Hop2 in mice eliminates homologous chromosome synapsis and disrupts double-strand break (DSB) repair through homologous recombination. HOP2 in vitro shows two distinctive activities: when it is incorporated into a HOP2–MND1 complex it stimulates DMC1 and RAD51 recombination activities and the purified HOP2 alone is proficient in promoting strand invasion. We observed that a fraction of Mnd1^−/− spermatocytes, which express HOP2 but apparently have inactive DMC1 and RAD51 due to lack of the HOP2–MND1 complex, exhibits a high level of chromosome synapsis and that most DSBs in these spermatocytes are repaired. This suggests that DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we show that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of the bases, and promotes the formation of a three-strand synaptic intermediate. However, HOP2 shows distinctive mechanistic signatures as a recombinase. Namely, HOP2-mediated strand exchange does not require ATP and, in contrast to DMC1, joint molecules formed by HOP2 are more sensitive to mismatches and are efficiently dissociated by RAD54. We propose that HOP2 may act as a recombinase with specific functions in meiosis.     

Overexpression of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA-binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the ‘recombination mediators’. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as ‘recombination co-mediators’. Defects in either recombination mediators or co-mediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic re-stabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51 expression.     

Homologous Recombination, but Not DNA Repair, Is Reduced in Vertebrate Cells Deficient in RAD52

Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the role of Rad52 in vertebrates, we generated RAD52^−/− mutants of the chicken B-cell line DT40. Surprisingly, RAD52^−/− cells were not hypersensitive to DNA damages induced by γ-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52^−/− cells as in wild-type cells. Targeted integration frequencies, however, were consistently reduced in RAD52^−/− cells, showing a clear role for Rad52 in genetic recombination. These findings reveal striking differences between S. cerevisiae and vertebrates in the functions of RAD51 and RAD52.     

The 9-1-1 DNA clamp is required for immunoglobulin gene conversion

Chicken DT40 cells deficient in the 9-1-1 checkpoint clamp exhibit hypersensitivity to a variety of DNA-damaging agents. Although recent work suggests that, in addition to its role in checkpoint activation, this complex may play a role in homologous recombination and translesion synthesis, the cause of this hypersensitivity has not been studied thoroughly. The immunoglobulin locus of DT40 cells allows monitoring of homologous recombination and translesion synthesis initiated by activation-induced deaminase (AID)-dependent abasic sites. We show that both the RAD9^−/− and RAD17^−/− mutants exhibit substantially reduced immunoglobulin gene conversion. However, the level of nontemplated immunoglobulin point mutation increased in these mutants, a finding that is reminiscent of the phenotype resulting from the loss of RAD51 paralogs or Brca2. This suggests that the 9-1-1 complex does not play a central role in translesion synthesis in this context. Despite reduced immunoglobulin gene conversion, the RAD9^−/− and RAD17^−/− cells do not exhibit a prominent defect in double-strand break-induced gene conversion or a sensitivity to camptothecin. This suggests that the roles of Rad9 and Rad17 may be confined to a subset of homologous recombination reactions initiated by replication-stalling lesions rather than those associated with double-strand break repair.     

Role of RAD51 in the repair of MuDR-induced double-strand breaks in maize (Zea mays L.)

Rates of Mu transposon insertions and excisions are both high in late somatic cells of maize. In contrast, although high rates of insertions are observed in germinal cells, germinal excisions are recovered only rarely. Plants doubly homozygous for deletion alleles of rad51A1 and rad51A2 do not encode functional RAD51 protein (RAD51⁻). Approximately 1% of the gametes from RAD51⁺ plants that carry the MuDR-insertion allele a1-m5216 include at least partial deletions of MuDR and the a1 gene. The structures of these deletions suggest they arise via the repair of MuDR-induced double-strand breaks via nonhomologous end joining. In RAD51⁻ plants these germinal deletions are recovered at rates that are at least 40-fold higher. These rates are not substantially affected by the presence or absence of an a1-containing homolog. Together, these findings indicate that in RAD51⁺ germinal cells MuDR-induced double-strand breaks (DSBs) are efficiently repaired via RAD51-directed homologous recombination with the sister chromatid. This suggests that RAD51⁻ plants may offer an efficient means to generate deletion alleles for functional genomic studies. Additionally, the high proportion of Mu-active, RAD51⁻ plants that exhibit severe developmental defects suggest that RAD51 plays a critical role in the repair of MuDR-induced DSBs early in vegetative development.     

Collaborative Action of Brca1 and CtIP in Elimination of Covalent Modifications from Double-Strand Breaks to Facilitate Subsequent Break Repair

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3′ and 5′ ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3′ single-strand overhang at “clean” DSBs, thus initiating homologous recombination (HR)–dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3′ single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIP^{S332A/−/−} cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP^+/−/− cells. Finally, CtIP^{S332A/−/−}BRCA1^−/− and CtIP^+/−/−BRCA1^−/− showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.     

The Cyclin-dependent Kinase Inhibitor Dacapo Promotes Genomic Stability during Premeiotic S Phase

The proper execution of premeiotic S phase is essential to both the maintenance of genomic integrity and accurate chromosome segregation during the meiotic divisions. However, the regulation of premeiotic S phase remains poorly defined in metazoa. Here, we identify the p21^Cip1/p27^Kip1/p57^Kip2-like cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) as a key regulator of premeiotic S phase and genomic stability during Drosophila oogenesis. In dap^−/− females, ovarian cysts enter the meiotic cycle with high levels of Cyclin E/cyclin-dependent kinase (Cdk)2 activity and accumulate DNA damage during the premeiotic S phase. High Cyclin E/Cdk2 activity inhibits the accumulation of the replication-licensing factor Doubleparked/Cdt1 (Dup/Cdt1). Accordingly, we find that dap^−/− ovarian cysts have low levels of Dup/Cdt1. Moreover, mutations in dup/cdt1 dominantly enhance the dap^−/− DNA damage phenotype. Importantly, the DNA damage observed in dap^−/− ovarian cysts is independent of the DNA double-strands breaks that initiate meiotic recombination. Together, our data suggest that the CKI Dap promotes the licensing of DNA replication origins for the premeiotic S phase by restricting Cdk activity in the early meiotic cycle. Finally, we report that dap^−/− ovarian cysts frequently undergo an extramitotic division before meiotic entry, indicating that Dap influences the timing of the mitotic/meiotic transition.     

Defective Repair of Oxidative Base Lesions by the DNA Glycosylase Nth1 Associates with Multiple Telomere Defects

Telomeres are chromosome end structures and are essential for maintenance of genome stability. Highly repetitive telomere sequences appear to be susceptible to oxidative stress-induced damage. Oxidation may therefore have a severe impact on telomere integrity and function. A wide spectrum of oxidative pyrimidine-derivatives has been reported, including thymine glycol (Tg), that are primarily removed by a DNA glycosylase, Endonuclease III-like protein 1 (Nth1). Here, we investigate the effect of Nth1 deficiency on telomere integrity in mice. Nth1 null (Nth1^−/−) mouse tissues and primary MEFs harbor higher levels of Endonuclease III-sensitive DNA lesions at telomeric repeats, in comparison to a non-telomeric locus. Furthermore, oxidative DNA damage induced by acute exposure to an oxidant is repaired slowly at telomeres in Nth1^−/− MEFs. Although telomere length is not affected in the hematopoietic tissues of Nth1^−/− adult mice, telomeres suffer from attrition and increased recombination and DNA damage foci formation in Nth1^−/− bone marrow cells that are stimulated ex vivo in the presence of 20% oxygen. Nth1 deficiency also enhances telomere fragility in mice. Lastly, in a telomerase null background, Nth1^−/− bone marrow cells undergo severe telomere loss at some chromosome ends and cell apoptosis upon replicative stress. These results suggest that Nth1 plays an important role in telomere maintenance and base repair against oxidative stress-induced base modifications. The fact that telomerase deficiency can exacerbate telomere shortening in Nth1 deficient mouse cells supports that base excision repair cooperates with telomerase to maintain telomere integrity.     

Focus: 50 Years of DNA Repair: The Yale Symposium Reports: BRCA2: One Small Step for DNA Repair,One Giant Protein Purified

DNA damage, malfunctions in DNA repair, and genomic instability are processes that intersect at the crossroads of carcinogenesis. Underscoring the importance of DNA repair in breast and ovarian tumorigenesis is the familial inherited cancer predisposition gene BRCA2. The role of BRCA2 in DNA double-strand break repair was first revealed based on its interaction with RAD51, a central player in homologous recombination. The RAD51 protein forms a nucleoprotein filament on single-stranded DNA, invades a DNA duplex, and initiates a search for homology. Once a homologous DNA sequence is found, the DNA is used as a template for the high-fidelity repair of the DNA break. Many of the biochemical features that allow BRCA2 to choreograph the activities of RAD51 have been elucidated and include: targeting RAD51 to single-stranded DNA while inhibiting binding to dsDNA, reducing the ATPase activity of RAD51, and facilitating the displacement of the single-strand DNA binding protein, Replication Protein A. These reinforcing activities of BRCA2 culminate in the correct positioning of RAD51 onto a processed DNA double-strand break and initiate its faithful repair by homologous recombination. In this review, I will address current biochemical data concerning the BRCA2 protein and highlight unanswered questions regarding BRCA2 function in homologous recombination and cancer.     

Analysis of Meiosis in SUN1 Deficient Mice Reveals a Distinct Role of SUN2 in Mammalian Meiotic LINC Complex Formation and Function

LINC complexes are evolutionarily conserved nuclear envelope bridges, composed of SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) domain proteins. They are crucial for nuclear positioning and nuclear shape determination, and also mediate nuclear envelope (NE) attachment of meiotic telomeres, essential for driving homolog synapsis and recombination. In mice, SUN1 and SUN2 are the only SUN domain proteins expressed during meiosis, sharing their localization with meiosis-specific KASH5. Recent studies have shown that loss of SUN1 severely interferes with meiotic processes. Absence of SUN1 provokes defective telomere attachment and causes infertility. Here, we report that meiotic telomere attachment is not entirely lost in mice deficient for SUN1, but numerous telomeres are still attached to the NE through SUN2/KASH5-LINC complexes. In Sun1^−/− meiocytes attached telomeres retained the capacity to form bouquet-like clusters. Furthermore, we could detect significant numbers of late meiotic recombination events in Sun1^−/− mice. Together, this indicates that even in the absence of SUN1 telomere attachment and their movement within the nuclear envelope per se can be functional.     

MUS81 Generates a Subset of MLH1-MLH3–Independent Crossovers in Mammalian Meiosis

Two eukaryotic pathways for processing double-strand breaks (DSBs) as crossovers have been described, one dependent on the MutL homologs Mlh1 and Mlh3, and the other on the structure-specific endonuclease Mus81. Mammalian MUS81 has been implicated in maintenance of genomic stability in somatic cells; however, little is known about its role during meiosis. Mus81-deficient mice were originally reported as being viable and fertile, with normal meiotic progression; however, a more detailed examination of meiotic progression in Mus81-null animals and WT controls reveals significant meiotic defects in the mutants. These include smaller testis size, a depletion of mature epididymal sperm, significantly upregulated accumulation of MLH1 on chromosomes from pachytene meiocytes in an interference-independent fashion, and a subset of meiotic DSBs that fail to be repaired. Interestingly, chiasmata numbers in spermatocytes from Mus81^−/− animals are normal, suggesting additional integrated mechanisms controlling the two distinct crossover pathways. This study is the first in-depth analysis of meiotic progression in Mus81-nullizygous mice, and our results implicate the MUS81 pathway as a regulator of crossover frequency and placement in mammals.     

Meiotic Recombination in Arabidopsis Is Catalysed by DMC1, with RAD51 Playing a Supporting Role

Recombination establishes the chiasmata that physically link pairs of homologous chromosomes in meiosis, ensuring their balanced segregation at the first meiotic division and generating genetic variation. The visible manifestation of genetic crossing-overs, chiasmata are the result of an intricate and tightly regulated process involving induction of DNA double-strand breaks and their repair through invasion of a homologous template DNA duplex, catalysed by RAD51 and DMC1 in most eukaryotes. We describe here a RAD51-GFP fusion protein that retains the ability to assemble at DNA breaks but has lost its DNA break repair capacity. This protein fully complements the meiotic chromosomal fragmentation and sterility of Arabidopsis rad51, but not rad51 dmc1 mutants. Even though DMC1 is the only active meiotic strand transfer protein in the absence of RAD51 catalytic activity, no effect on genetic map distance was observed in complemented rad51 plants. The presence of inactive RAD51 nucleofilaments is thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1. Our data demonstrate that RAD51 plays a supporting role for DMC1 in meiotic recombination in the flowering plant, Arabidopsis.     

DNA damage response in microcephaly development of MCPH1 mouse model

MCPH1 encodes BRCT-containing protein MCPH1/Microcephalin/BRIT1, mutations of which in humans cause autosomal recessive disorder primary microcephaly type 1 (MCPH1), characterized by a congenital reduction of brain size particularly in the cerebral cortex. We have shown previously that a deletion of Mcph1 in mice results in microcephaly because of a premature switch from symmetric to asymmetric division of the neuroprogenitors, which is regulated by MCPH1's function in the centrosome. Because MCPH1 has been implicated in ATM and ATR-mediated DNA damage response (DDR) and defective DDR is often associated with neurodevelopmental diseases, we wonder whether the DDR-related function of MCPH1 prevents microcephaly. Here, we show that a deletion of Mcph1 results in a specific reduction of the cerebral cortex at birth, which is persistent through life. Due to an effect on premature neurogenic production, Mcph1-deficient progenitors give rise to a high level of early-born neurons that form deep layers (IV–VI), while generate less late-born neurons that form a thinner outer layer (II–III) of the cortex. However, neuronal migration seems to be unaffected by Mcph1 deletion. Ionizing radiation (IR) induces a massive apoptosis in the Mcph1-null neocortex and also embryonic lethality. Finally, Mcph1 deletion compromises homologous recombination repair and increases genomic instability. Altogether, our data suggest that MCPH1 ensures proper neuroprogenitor expansion and differentiation not only through its function in the centrosome, but also in the DDR.     

Mouse TEX15 is essential for DNA double-strand break repair and chromosomal synapsis during male meiosis

During meiosis, homologous chromosomes undergo synapsis and recombination. We identify TEX15 as a novel protein that is required for chromosomal synapsis and meiotic recombination. Loss of TEX15 function in mice causes early meiotic arrest in males but not in females. Specifically, TEX15-deficient spermatocytes exhibit a failure in chromosomal synapsis. In mutant spermatocytes, DNA double-strand breaks (DSBs) are formed, but localization of the recombination proteins RAD51 and DMC1 to meiotic chromosomes is severely impaired. Based on these data, we propose that TEX15 regulates the loading of DNA repair proteins onto sites of DSBs and, thus, its absence causes a failure in meiotic recombination.     

Hyper-resistance of meiotic cells to radiation due to a strong expression of a single recA-like gene in Caenorhabditis elegans

Sensitivity of meiotic cells to DNA damaging agents is little understood. We have demonstrated that the meiotic pachytene nuclei in the Caenorhabditis elegans gonad are hyper-resistant to X-ray irradiation, but not to UV irradiation, whereas the early embryonic cells after fertilization and the full grown oocytes are not. The Ce-rdh-1 gene [RAD51, DMC1 (LIM15), homolog 1 or Ce-rad-51], which is essential for the meiotic recombination, is the only bacterial recA-like gene in the nematode genome, and is strongly expressed in the meiotic cells. Following silencing of the Ce-rdh-1 gene by RNA interference, the meiotic cells become more sensitive to X-ray irradiation than the early embryonic cells. This is the first report that meiotic cells are hyper-resistant to DNA strand breaks due to the high level of expression of the enzyme(s) involved in meiotic homologous recombination.     

ALKBH1 Is Dispensable for Abasic Site Cleavage during Base Excision Repair and Class Switch Recombination

Potential roles of the abasic site lyase activity associated with AlkB homolog 1 (ALKBH1) were assessed by studies focusing on the two cellular processes that create abasic sites as intermediates: base excision repair and class switch recombination. Alkbh1^−/− pups (lacking exon 3) were born at a lower than expected frequency from heterozygous parents, suggesting a reduced survival rate and non-Mendelian inheritance, and they exhibited a gender bias in favor of males (70% males and 30% females). To study ALKBH1’s potential involvement in DNA repair, fibroblasts were isolated from Alkbh1^−/− mice, spontaneously immortalized and tested for resistance to DNA damaging agents. Alkbh1^−/− and isogenic cells expressing hALKBH1 showed no difference in survival to the DNA damaging agents methyl-methionine sulfate or H₂O₂. This result indicates that ALKBH1 does not play a major role in the base excision repair pathway. To assess ALKBH1’s role in class switch recombination, splenic B cells were isolated from Alkbh1^−/− and Alkbh1^+/+ mice and subjected to switching from IgM to IgG1. No differences were found in IgG1 switching, suggesting that Alkbh1 is not involved in class switch recombination of the immunoglobulin heavy chain during B lymphocyte activation.     

MCPH1/BRIT1 cooperates with E2F1 in the activation of checkpoint, DNA repair and apoptosis

Microcephalin (MCPH1) has a crucial role in the DNA damage response by promoting the expression of Checkpoint kinase 1 (CHK1) and Breast cancer susceptibility gene 1 (BRCA1); however, the mechanism of this regulation remains unclear. Here, we show that MCPH1 regulates CHK1 and BRCA1 through the interaction with E2F1 on the promoters of both genes. MCPH1 also regulates other E2F target genes involved in DNA repair and apoptosis such as RAD51, DDB2, TOPBP1, p73 and caspases. MCPH1 interacts with E2F1 on the p73 promoter, and regulates p73 induction and E2F1-induced apoptosis as a result of DNA damage. MCPH1 forms oligomers through the second and third BRCT domains. An MCPH1 mutant containing only its oligomerization domain has a dominant-negative role by blocking MCPH1 binding to E2F1. It also inhibits p73 induction in DNA damage and E2F1-dependent apoptosis. Taken together, MCPH1 cooperates with E2F1 to regulate genes involved in DNA repair, checkpoint and apoptosis, and might participate in the maintenance of genomic integrity.