DNA helicases are required for virtually every aspect of DNA metabolism, including replication, repair, recombination and transcription. A comprehensive understanding of these essential biochemical processes requires knowledge of the enzymatic mechanisms of DNA helicases. The goal of this research is to use new methods that we have developed to investigate helicase-catalyzed DNA unwinding in greater detail than has previously been possible. We will study two viral helicases as representatives of a broad functional range of activities. SV40 T antigen, from the SV40 virus, is a replicative helicase that functions as a double hexamer and unwinds DNA in a highly processive manner. Dda, from bacteriophage T4, is involved in replication initiation and recombination, functions as a monomer or dimer, and unwinds DNA in a distributive manner. DNA unwinding and translocation are not well understood, due to inadequate methods to measure these processes. We have developed a pre-steady state assay for DNA unwinding in which the observed burst amplitude will be evaluated as a function of enzyme and substrate concentration. This assay will serve as an active site titration and will be valuable for relating unwinding to ATP hydrolysis. Two methods will be used to measure the number of base pairs unwound in a single catalytic cycle or """"""""step size"""""""": 1) Unwinding rates at unique base pair positions will be determined by chemical modification of newly formed single-stranded DNA using potassium permanganate, and 2) A fluorescence spectroscopic method will be utilized to measure unwinding rates at unique positions by incorporating fluorescent nucleotide analogs into specific sites in DNA substrates. Translocation on ssDNA will be investigated by measuring the rate of helicase-catalyzed displacement of proteins bound to the ends of oligonucleotides. To determine whether there exists a bias in direction of movement on single-stranded DNA, the protein blocks will be placed on either end of the oligonucleotide. The functional significance of dsDNA during the unwinding reaction will be probed using chemically modified DNA substrates to determine the degree to which helicases interact with the duplex during the reaction cycle. The new methods utilized here will define the biochemical mechanisms of DNA helicases in a quantitative manner that will be useful for evaluating the role of helicases in DNA metabolism and disease.
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