of work: Chromosomal double-strand breaks (DSBs) pose a significant and immediate danger to genome integrity and cell survival. DSBs can arise through the action of exogenous agents (radiation and chemicals), endogenous causes (e.g., collapsed replication forks) and in the course of developmental programs (Ig recombination and class switching, and meiosis). DSBs are repaired by either nonhomologous end joining or homologous recombination (HR). Both pathways are equally important in most organisms, including mammals (reviewed in Sonoda et al. (2006) DNA Repair 5, 1021)). HR is common to all forms of life and is a multistep process involving many gene products. In eukaryotes, the biologically most important form of recombination is the exchange of genetic information between homologous chromosomes (homologs) in meiosis (reviewed in Gerton and Hawley (2005) Nature Rev. Genet. 6, 477). Meiotic recombination is the central phenomenon in the genetics of eukaryotes, ensures the proper segregation of chromosomes at the first division of meiosis (prevents non-disjunction) and is the main force shaping the evolution of genomes. In all organisms, homologous recombination is inextricably related to DNA repair and replication. Rad51 and the meiosis-specific Dmc1 protein, both homologues of bacterial RecA (the prototypical homologous recombination protein), have been identified in most eukaryotes including man and mouse, and homozygous Rad51 -/- mouse ES cells are not viable. Thus, the study of RecA homologues should yield insights into not only homologous recombination but also the regulation of gene stability and cell proliferation. In meiosis in all organisms, including mammals (Romanienko and Camerini-Otero (2000) Mol. Cell 6, 975), Spo11, a type II-like topoisomerase, cleaves the chromosomal DNA at many sites (a couple of hundred or more in mammals) in each and every nucleus (reviewed in Keeney and Neale (2006) Biochem. Soc. Trans. 34, 523). In mammals there are more endogenous DSBs in meiotic nuclei than in any somatic nuclei in the life of an organism, by a factor of at least 100. Hence the entire arsenal of the HR machinery invoked in somatic cells plus additional meiosis-specific proteins are required for the efficient repair of DSBs in meiosis. These include p53, Brca1, Brca2, Rad51, Dmc1, Atm, Atr, DNA-PKcs, Chk2, Nbsl, Mre11, Rpa, Blm, etc. Spo11 is required for meiotic chromosomal synapsis in S. cerevisae. Surprisingly, Spo11 homologues are dispensable for synapsis in C. elegans and D. melanogaster yet required for meiotic recombination. We have generated a SPO11 mouse knock-out to investigate the biological function of this gene in mammals. Disruption of mouse SPO11 results in infertility. Spermatocytes arrest prior to pachytene with little or no homologous synapsis and undergo apoptosis (Romanienko and Camerini-Otero (2000) Mol. Cell 6, 975). Surprisingly, Spo11 heterozygosity rescues the meiotic prophase arrest seen in mice lacking the Atm (ataxia telangiectasia, mutated ) double-strand break signaling protein (Bellani, Romanienko, Cairatti and Camerini-Otero (2005) J. Cell Science 118, 3233). Most recently, we have used a rabbit monoclonal antibody to the Spo11 protein to confirm that in fact most of the Spo11 protein is expressed late in prophase I and is found in the cytoplasm. Furthermore, we have been able to show that it is the alpha-isoform that is most abudantly expressed and that is expressed late. Finally, we have reexamined the expression profiles of the two major splicing isoforms of Spo11, Spo11alpha (exon 2 skipped) and Spo11beta found in both mice and humans. Our data argues for a major role for Spo11beta in introducing the breaks that initiate meiotic recombination. Furthermore, we find that Spo11alpha is the form that is most expressed but mainly after the DSBs are introduced and this expression is seen in both spermatocytes and oocytes. We propose a role for Spo11alpha in mid- to late prophase, presumably acting as a topoisomerase, in both male and female meiocytes. In order to understand the role of SPO11 alpha;, we have generated a transgenic mouse carrying a BAC with a modified Spo11 locus inserted ectopically into the genome. We were able to detect over-expression of SPO11 αin a wild type mouse carrying the transgene and found that over-expression of SPO11 αdoes not affect meiotic progression. We found that even though SPO11 αis expressed early in a Spo11-/- Spo11α+/- transgenic mouse, it fails to rescue the meiotic arrest characteristic of the Spo11 knockout mouse. This supports the notion that the SPO11 βisoform is responsible for introducing the breaks. At the moment we are trying to address whether SPO11αis able to rescue the KO phenotype after introducing DSBs exogenously. We plan to generate a knock-in mouse where the endogenous Spo11 locus would be replaced by the construction expressing only SPO11 α. Recently, in a collaboration with the labs of Maria Jasin and Scott Keeney at Sloan Ketterin Cancer Center, we have been examining whether Spo11alpha can complement a sterile transgenic mouse carrying Spo11beta. Finally and most recently, in a collaboration with the lab of Galina Petukhova at USUHS we have examined how a hypomorphic allele of Spo11 affects the genome-wide distribution of double-strand breaks in a mouse. The results have indicated that in such a mouse there is suppression of genetic recombination in the pseudoautosomal and subtelomeric regions.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$439,616
Indirect Cost
City
State
Country
Zip Code
Smagulova, Fatima; Brick, Kevin; Pu, Yongmei et al. (2013) Suppression of genetic recombination in the pseudoautosomal region and at subtelomeres in mice with a hypomorphic Spo11 allele. BMC Genomics 14:493
Kouznetsova, Anna; Wang, Hong; Bellani, Marina et al. (2009) BRCA1-mediated chromatin silencing is limited to oocytes with a small number of asynapsed chromosomes. J Cell Sci 122:2446-52