of work: In order to dissect the biochemical steps involved in genetic recombination we have chosen to focus on a key early step(s): homologous pairing and strand exchange between homologous parental DNAs. A fundamental problem in homologous recombination is how the search for homology between the two DNAs is carried out. In all current models a homologous recombination protein, such as the prototypical E. coli RecA protein, loads onto a single-strand DNA generated from one duplex DNA and scans another duplex to form a synaptic (pairing) complex. Eventually, DNA strands are exchanged and a new heteroduplex is formed. While homologous pairing and strand exchange are the earliest contacts between two parental DNAs mediated by RecA and its eukaryotic homologues, Rad51 and Dmc1, homologous recombination is initiated by DNA double-strand breaks (DSBs). The protein that catalyzes DSB formation in meiosis in the budding yeast, Saccharomyces cerevisae, is the product of the SPO11 gene. Disruption of this gene results in meiotic arrest, spore lethality and a lack of meiotic recombination. Spo11 homologues have been identified in other eukaryotes and archaebacteria resulting in the identification of a new family of proteins related to DNA topoisomerase IIs. We have identified and cloned Spo11 homologues in fruit flies, mouse and man. In mouse and man northern blot analysis revealed testis-specific expression of SPO11, but RT-PCR revealed expression is somatic tissues as well. Both the mouse and human transcripts undergo alternative splicing. Chromosome localization was performed for both mouse and human SPO11, and the human gene was localized to chromosome 20q13.2-13.3, a region amplified in some breast and ovarian cancers. Finally, using affinity-purified antibodies to the mouse protein we have visualized Spo11 as individual foci as early as leptotene, the stage in meiosis I when DSBs are generated, and later in zygotene and pachytene of the meiotic prophase in spermatocytes. In pachytene Spo11 is foundonly in those areas where the chromosomes are fully synapsed. 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 synapsis and undergo apoptosis. We did not detect Rad51/Dmc1 foci in meiotic chromosome spreads, indicating DSBs are not formed. Cisplatin-induced DSBs restored Rad51/Dmc1 foci and promoted synapsis. Other mouse mutants that arrest during meiotic prophase (Atm -/-, Dmc1 -/-, mei1, Morc -/-) showed altered Spo11 protein localization and expression. In particular, an alternatively spliced transcript is almost absent in testes from these mutants and very little Spo11 is seen during pachytene. We speculate that there is an additional role for Spo11, after it generates DSBs, in synapsis. Most recently, we have analyzed the meiotic phenotypes of Spo11 Atm and Spo11 p53 double knockouts in order to unravel how DSBs are sensed during meiosis in mammals. Also we have made Spo11 knock-ins where the catalytic site has been altered but small amounts of the total protein are being made. The phenotype of this mouse is now under investigation. In all organisms, homologous recombination is inextricably related to DNA repair and replication, hence cell proliferation and its control. For example, in E. coli, RecA, the prototypical homologous recombination protein, is directly responsible for turning on the SOS response to genotoxic damage. The RecA-ssDNA- ATP filament, the active form of RecA, acts as a co-protease in the auto-catalytic digestion of the LexA repressor. Much less is known about how the SOS response is extinguished. DinI is the product of a damage-inducible, LexA-controlled gene. Previous work has shown that when this gene is over-expressed in mitomycin C-treated cells it prevent the cleavage of LexA and UmuD proteins. Furthermore, in vitro DinI prevents the cleavage of UmuD promoted by the RecA-ssDNA co-filament. Our experiments with purified RecA and DinI have revealed that they interact directly. While DinI does not bind to DNA it releases ssDNA from the active RecA-ssDNA co-filament. Furthermore, the C-terminal portion of DinI interacts with the DNA binding and homologous pairing of RecA, loop L2. In collaboration with Ad Bax and Ben Ramirez of the Laboratory of Chemical Physics of NIDDK, we have shown that this region of DinI has a structure that appears as a DNA mimic. Such a DNA mimic acts as a competitor for DNA on RecA. We have now identified several other proteins that have such domains that resemble this DNA mimic. We are now investigating whether these proteins bind to DNA-binding proteins in the same manner that DinI binds to RecA. Many of these proteins are of eukaryotic origin. Finally, we have used whole-genome cDNA macroarrays were used to analyse changes in the levels of gene expression of all E. coli ORFs after treatment with mitomycin C (MMC). Several experiments, which differ in the mode of MMC treatment, were performed, and expression profiles of E. coli cells at different time points after the addition of the DNA damaging agent were analyzed. As a whole, these experiments consist of 16 different hybridizations corresponding to about 70,000 individual data points. Around 5-10% of all genes show significant changes in their level of expression. As shown before, the expression level of several LexA-regulated genes was increased after DNA damage. On the other hand, most of those genes that show significant changes in their level of expression have not been shown previously to be inducible or repressed in the process of DNA repair. An attempt was made to classify all genes based on their responses to DNA damage. Using cluster analysis of the gene expression data it is possible to divide all the genes into at least 12 different clusters. We also compared gene expression data with biochemical pathways in which corresponding genes are involved and positional information. The analysis has allowed us to identify new candidate genes in DNA repair.
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