GENE EXPRESSION AND HUMAN GENETICS

Diabetes, Digestive, Kidney Diseases

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Abstract

Summary 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 only in pachytene of meiosis I in spermatocytes and only in those areas where the chromosomes are fully synapsed.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. - Recombination, DNA structure, meiosis, ATP hydrolysis, allostery, double-strand breaks, RecA, Spo11