Analysis of regulation and function of the BLM helicase

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DESCRIPTION (provided by applicant): Genomic instability is a condition of a cell in which mutations occur at a frequency greater than normal. Such a condition can arise from defects in DNA replication, DNA repair, or the cell's damage control machinery. A primary defect in one of these cellular systems characterizes the rare autosomal recessive disorder Bloom syndrome's (BS). The BS gene, BLM, encodes a RecQ DNA helicase. Absence of the BLM helicase from a cell causes a striking genomic instability that includes both chromosome breakage and excessive chromatid exchange, in particular, sister chromatid exchange (SCE). The hypermutability and hyperrecombinability characteristic of BS cells doubtless explains the striking cancer predisposition of BS persons; however, the normal function(s) of the BLM helicase and the molecular mechanisms by which the protein's absence causes genomic instability in BS cells are not understood. The BLM helicase is present in the cell in a diffuse, microspeckled form and in characteristic nuclear dots that we have identified as the PML nuclear bodies (PML-NBs). Like PML, BLM is covalently modified by a ubiquitin like molecule, SUMO-1 and SUMO-2, which we hypothesize functions to localize BLM to the PML-NBs. Aim 1 of the proposal is to identify and characterize the regions of BLM that mediate localization to the PML-NBs. We experimentally introduce different mutations (deletions, insertions, and amino acid substitutions) into a GFP-BLM expression construct and express the mutant proteins in various cells, including HeLa and BS cells. Then we assay the functional consequences of the mutations on localization to the PML-NBs, BLM helicase activity,and complementation of the BS cellular phenotype by the SCE assay. Aim 2 is to determine whether SUMO modification of BLM causes localization to the PML-NBs by performing a mutational analysis similar to that in Aim I and using an in vitro modification assay to map interactions and SUMO-sites. We analyze the functional consequences of modification on BLM localization, helicase activity, and complementation of the BS cellular phenotype. In Aim 3, we investigate the function of the BLM-Topoisomerase III alpha (Topo III) complex. We perform a mutational analysis of the domain of BLM that interacts with Topo III by the yeast two-hybrid screen. Then, we determine the functional consequences of these mutations by measuring the ability of the proteins to interact, the activity of the complex in helicase and topoisomerase assays, and its action in BS cell compimentation. Additionally, we measure the effect of SUMO modification on BLM Topo III interaction and the complexes enzymatic activity. In Aim 4, we analyze the domain structure of BLM and the role of oligomerization in helicase activity and BS cell complementation.