Studies are proposed to analyze the human and mouse homologues of the recently described Drosophila protein barren. In Drosophila, barren is required for chromatic arm separation and has been shown to interact with topo II and to modulate its activity in vitro. Dr. Belmont and his colleagues intend to test the hypothesis that barren function is conserved and that its activity in mammalian chromatid arm separation is similar to that observed in Drosophila. They have partially characterized the gene encoding human barren (BRRN-1) and have identified other members of a putative barren gene family in human and mouse genomes. The investigators propose to employ several strategies to better understand the function of barren-1 and its mechanism of action in human cells. The first approach will examine barren-1 expression and subcellular localization in the cell cycle. They will also analyze barren-1 expression in Roberts syndrome cells, a human mutation which appears to involve a specific defect in chromatid arm separation. They will then evaluate potential barren interaction with topoisomerase II, and mammalian homologues of the SMC (structural maintenance chromosomes) proteins, XCAP-C and XCAP-E. The final strategy will employ targeted inactivation of mouse mbrrn-1 in cultured ES cells. Both simple and inducible inactivation constructs will be tested. Depending on the outcome of these experiments production heterozygous and homozygous mutant mice will be attempted. Similar mutants of the mouse lodestar-like helicase gene will also be evaluated. In Drosophila lodestar mutants appear to affect chromatid arm separation. Genetic interaction and effects on barren expression will be examined. The proposed studies should yield valuable information on the regulation of topo II and its participation in the anaphase process of mammalian cells. Elucidation of these events has broader implications for understanding chromosome non-disjunction and other human disorders associated with chromosome instability.
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