DNA helicases are required for virtually every aspect of DNA metabolism, including replication, repair, recombination and transcription. These enzymes are ubiquitous, having been identified in viruses, prokaryotes, and eukaryotes. Diseases such as xeroderma pigmentosum, Cockayne's syndrome, Bloom's syndrome, and Werner's syndrome, have been linked to defects in specific genes coding for DNA helicases. A comprehensive description of these essential biochemical processes requires detailed understanding of helicase mechanisms. For some helicases, enzymatic activity is dependent on the number of helicase molecules that are bound onto the DNA. This mechanism is referred to as the cooperative inchworm model n the case of Dda helicase or the functional cooperativity model in the case of NS3h helicase. Each individual helicase is thought to function via an inchworm mode of translocation, but multiple molecules bound to a single substrate exhibit greater activity. Cooperativity can result through structural or functional interactions. The goal of this R24 research proposal is to bring together investigators to develop methods that will allow direct visualization of helicase movement at the single molecule level in order to test the cooperative inchworm mechanism and lay the groundwork for studying multiprotein complexes on DNA. Helicases will be labeled with highly fluorescent nanocrystals, thereby enabling individual or groups of molecules to be observed directly. Direct visualization of single and multiple helicases during unwinding of double stranded DNA (specific aim 1) and translocation on single stranded DNA (specific aim 2) willl be measured. Three helicases that have unique properties will be examined. Tral is a highly processive helicase whereas Dda and NS3h are poorly processive helicases. Dda and NS3h are proposed to function cooperatively whereas Tral is proposed to function as a monomer. This research proposal is a direct response to the program announcement PA 03-127, """"""""INTEGRATIVE AND COLLABORATIVE APPROACHES TO RESEARCH"""""""" which is to facilitate collaboration between NIH-funded investigators who use different technologies to study a common problem. ? ? ?
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| Tahmaseb, Kambiz; Matson, Steven W (2010) Rapid purification of helicase proteins and in vitro analysis of helicase activity. Methods 51:322-8 |
