The proposed research is to conduct in vitro studies of the mechanisms by which duplex DNA is catalytically unwound by two classes of DNA binding proteins: helicases and helix destabilizing proteins (HDP). In particular, the E. coli coded Rep and Helicase II proteins (helicases) and the E. coli coded Single Strand Binding protein, SSB, (HDP) will be studied. The helicases catalyze the ATP dependent, unidirectional unwinding of duplex DNA, a process which requires translocation of the protein along DNA and is fundamental to DNA replication. Physical-biochemical techniques will be used to quantitatively investigate the equilibrium and kinetic binding properties of the purified proteins to DNA as a function of solution variables, with particular emphasis on the effects of low molecular weight ions (NaC1, MgC12, spermidine). The basis for the cooperativity in SSB-single stranded DNA binding and its dramatic dependence on salt concentration will also be studied. In parallel with these studies, the mechanisms of DNA unwinding catalyzed by Rep and Helicase II, and the role of SSB in this process, will be investigated, focusing on the rates, processivity, and role of ATP. The molecular aspects of the translocation of protein along DNA, which is fundamental to many cellular processes, will also be probed. The methods used to study the protein-DNA interactions include analytical sedimentaiton, gel electrophoresis, fluorescence and absorbance spectroscopy, affinity and gel chromatography and stopped-flow kinetics. The DNA unwinding reaction will be studied in the absence of DNA synthesis and other replication proteins, hence providing a simple system to probe the molecular aspects of unwinding and tranlocation. These studies are specifically directed to understand the helicase catalyzed DNA unwinding reaction and the mechanism of the ATP driven helicase translocation, however they will also reveal thermodynamic details which will increase our general knowledge of the factors which stabilize protein-nucleic acid interactions. Furthermore, the mechanistic information obtained from studies of the kinetics of these protein-DNA interactions should be useful in studies of other proteins which must also translocate along DNA (driven thermally or by hydrolysis of ATP); e.g. DNA and RNA polymerases. Since DNA replication is fundamental to cell growth, an understanding of its detals will undoubtedly increase our chances for determining how it malfunctions in diseases such as cancer.
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