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 at 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. 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 found only 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 homologous 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. We speculate that there is an additional role for Spo11, after it generates DSBs, in synapsis. Recently, we have been conducting DNA microarray experiments to determine those meiotically expressed genes whose expression is modified by a DSB. We have been comparing RNA populations from testis harvested from wild type, Spo11 knock-out and irradiated mice of both types. In young mice, before degenerative changes have set in as a result of the apoptosis seen in the knockouts, there are only a few dozen genes that are differentially expressed in Spo11 -/- compared to wild type. Among the genes most affected are the Hop2 and Mnd1 genes. These are homologues of genes that affect homologous pairing in yeast. We have generated a knockout of the Hop2 gene in the mouse. Its meiotic phenotype shows a profound meiotic arrest that is unlike any seen previously. Unlike most knockouts with a meiotic phenotype these mice show no synapsis of any kind. That is, whereas most knockouts, show some willy-nilly non-homologous synapsis, spermatocytes from these mice are arrested without almost any synapsis. The chromosomes are somewhat compacted and appear normally decorated with both Rad51 and Dmc1, as if they are on the cusp of synapsis but fail to proceed forward. We are now in the process of examining the biochemical properties of the Hop2 protein to determine how they may help explain this very unusual phenotype. 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. Recently we (in collaboration with Ben Ramirez and Ad Bax of LCP) reported a model for the abrogation of the SOS response by this SOS proein and proposed that a negatively charged helix on the C-terminus of DinI mimics DNA in its interaction with RecA, effectively short-circuiting the SOS response. 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 arrays were used to analyze 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. Of the 400 or so upregulated genes about 100 were poorly annotated or not annotated at all. Of these 100 we have selected about 50 that encode for proteins of modest size, are not clearly membrane proteins and show some evolutionary conservation. We have made gene deletion strains for most of these genes and are now studying their phenotypes, both with regard to DNA metabolism (in collaboration with Sue Lovett at Brandeis) and general intermediate metabolism using the Biolog phenotypic arrays. In addition, we have initiated a structural genomics project (in collaboration with Galya Obmolova, Alex Teplyakov and Gary Gilliland at CARB) to determine the structure of as many as possible of the protein products of these 50 genes.
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