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Rapid kinetics study of the nuclease activity of RecBCD enzyme from Escherichia coli

dc.contributor.advisorJulin, Douglasen_US
dc.contributor.authorGHATAK, ARCHANAen_US
dc.description.abstractRecBCD enzyme from Escherichia coli is involved in homologous recombination and repair of bacterial DNA, and in defending the cell against foreign DNA. This enzyme has multiple functions - it is an ATP-stimulated ssDNA endonuclease, an ATP-dependent ssDNA and dsDNA exonuclease and a DNA helicase. Here we investigated the kinetics of the exonuclease reaction of RecBCD. We purified the enzyme from E. coli by using conventional adsorption chromatography. The exonuclease function was studied using a rapid quench-flow (RQF) instrument designed by Kintek Corporation. Short single-stranded DNA oligomers were labeled with 32P ATP at the 5 end and used as substrates. Our goal was to study the reaction products as the enzyme travels along the DNA. From earlier studies it is known that the enzyme binds at the end of a double-stranded DNA, unwinds and cleaves the DNA as it moves. This enzymatic movement can be very fast, in the range of 500−1000 base-pairs per second. We carried out exonuclease reactions using the RQF instrument for time periods ranging from 10 msec to 2 sec. The time courses of appearance and change of the amounts of the labeled products displayed in sequencing gels were analyzed using a Phosphorimager and associated software. A kinetic model for the enzyme was derived by analyzing the time course data with Kinteksim, a simulation program. We were able to identify the reaction products of the single turnover exonuclease reactions. For the first time we were able to show that the enzyme does not cleave a DNA substrate at every nucleotide even in conditions where it acted as a potent exonuclease. Rather, our results suggested that the enzyme moves along the DNA and cleaves in steps of 2-4 nucleotides. The enzyme concentration used in our study was almost 10 times more than the DNA concentration in order to make sure that all DNA molecules were bound to enzyme. Still not all DNA molecules participated in the nuclease reaction in the single turnover reaction time period, suggesting that some of the DNA enzyme complexes were inactive with respect to the nuclease activity. The rate of translocation of the enzyme along the DNA varied with the change of concentration of ATP in the reaction. However, the products were the same at several ATP concentrations. Our results indicate the presence of at least two ATP dependent steps after the reaction is started and before the first nuclease product appears. These steps are proposed to represent the translocation of the DNA end from the binding site to the nuclease active site. Also, our results suggest that the rate of translocation at each ATP concentration is similar to the unwinding rate of a double stranded DNA at the same ATP concentration [determined by Lucius, A. L. & Lohman, T. M. (2004) J Mol Biol 339, 751-771.]en_US
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dc.titleRapid kinetics study of the nuclease activity of RecBCD enzyme from Escherichia colien_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.subject.pqcontrolledChemistry, Biochemistryen_US

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