Loss of genome stability is associated with a number of human diseases that predispose patients to cancer. The RecQ family of DNA helicases is a key player in the maintenance of genome stability. Although these enzymes participate in homologous recombination to repair double-stranded DNA breaks and stalled replication forks, a clear understanding of the mechanism of DNA repair by these enzymes is lacking. BLM, a RecQ helicase responsible for Bloom's Syndrome, is widely conserved across eukaryotic species where it binds the Top3 and Rmi1 subunits to form a complex referred to as BTR. In the yeast model system this complex is referred to as STR (Sgs1-Top3-Rmi1). This application proposes to use the yeast model system to determine the enzymatic properties of the N-terminal non-helicase domains of RecQ helicases. A newly identified domain (the SSD) is essential for Sgs1 function and displays DNA strand swapping (SS) activity. SS activity is conserved in human BLM, WRN, and RecQ4 and in all cases the SSDs are associated with coiled-coil multimerization domains.
In Aim 1 we will test the hypothesis that the SSD cooperates with Top3-Rmi1 or the helicase domain to promote DNA unwinding and DNA strand passage using model substrates.
In Aim 2 we will examine the physiological role of the SSD by asking whether it is required for STR activity in vivo. Sgs1 proteins lacking the SSD will be assayed for effects on gene conversion and crossing- over.
In Aim 3 we will characterize the multimerization domain and determine whether it is required to complement defects in BLM-/- cells. Because SS activity is conserved, but the SSDs show little or no amino acid sequence similarity, we will determine whether the yeast and human SSDs are structurally similar.
In Aim 4 we will extend our studies of the non-helicase domain of Sgs1 to characterize a second domain required for Sgs1 function. The domain will be characterized biochemically by searching for DNA binding activity and by testing whether comparable residues from human BLM function in yeast. Successful completion of these experiments will reveal the function of non-helicase domains of the RecQ family and the potential substrates of BTR/STR during DNA repair in vivo.
Genomic instability is one of the most common characteristics of cancer cells and may be a precipitating factor in generating the transformed phenotype. BLM is a member of RecQ family of DNA helicases that acts to insure genome stability in response to DNA damage. This project seeks to determine the enzyme mechanism that BLM uses to suppress inappropriate recombination using yeast as a model system. The project will exploit well-known features of this model system to determine the role of a newly-identified enzyme activity and how it cooperates with other activities in the BLM protein complex. Thus, this research will provide new understanding about the biological pathways that maintain genome stability.
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