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Title

Transient-State Kinetic Studies of Escherichia coli UvrD Monomer Translocation along Single-Stranded DNA

Date of Award

Spring 5-15-2010

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

E. coli UvrD is a DNA helicase that functions in a variety of DNA metabolic processes. I investigated the ssDNA translocation mechanism of the UvrD monomer, a required component of the dimeric UvrD helicase. UvrD is a superfamily I DNA helicase/ translocase that unwinds DNA and translocates along ssDNA, 3’ to 5’, using an ATPdependent mechanism. Using transient-state kinetic approaches I studied UvrD monomer translocation by monitoring three features of the translocation process as a function of ssDNA length: 1.) Arrival of the UvrD monomer at the 5’-end of the ssDNA; 2.) ATP hydrolysis during UvrD translocation; and 3.) UvrD dissociation from the ssDNA during translocation. Global analysis of all three time courses to an n-step sequential mechanism provides estimates of the translocation rate, processivity, kinetic step size, ATP coupling stoichiometry, and UvrD dissociation rate constants from the ssDNA, placing constraints on possible translocation mechanisms. I studied UvrD monomer translocation over a range of ATP and salt concentrations, in the presence of duplex DNA structures, and on different ssDNA base compositions to determine the UvrD monomer translocation mechanism and how it is affected by changes in the DNA substrate.

I found that the UvrD monomer translocation mechanism along ssDNA is consistent with an inchworm stepping model where the UvrD monomer hydrolyzes one ATP per nucleotide translocated (10 mM Tris-HCl, pH 8.3, 20 mM NaCl, 20% (v/v) glycerol, and 2 mM MgCl2 at 25°C). This ATP coupling stoichiometry did not change over a range of ATP concentrations (10-500 µM) spanning above and below the Michaelis-Menten constant for ATP binding to UvrD (33 µM), suggesting tight coupling between ATP hydrolysis and 3’to 5’ translocation along the ssDNA. The ATP coupling stoichiometry suggests a 1-nt translocation step size; however, the translocation kinetic step size, the average distance translocated between consecutive rate-limiting translocation steps, is larger (~4 nts at saturating [ATP]) and it changes with both ATP and salt concentration. The larger kinetic step size and the small ATP coupling stoichiometry could reflect a discontinuous stepping mechanism where the UvrD monomer translocates in rapid, 1-nt steps each hydrolyzing one ATP followed by a slower step that limits the overall rate of translocation; however, the ATP dependent change in the kinetic step size is not entirely consistent with this model. Rather the ATP and salt dependence changes in the translocation kinetic step size, suggest the presence of kinetically different populations of UvrD on the ssDNA.

Translocation experiments on partial DNA duplex substrates with a 5’ flanking ssDNA tail also indicate the presence of kinetically different populations of UvrD, where UvrD that specifically initiates translocation from the ss/ds junction has a significantly smaller kinetic step size than UvrD initiating translocation from internal sites of the ssDNA tail. Interestingly, UvrD monomer specificity for binding to a 5’-ss/dsDNA junction, which orients UvrD to translocate away from the junction, suggest such iiijunctions may serve to specifically load the UvrD monomer on DNA where the ssDNA translocase activity of the monomer is required. This could be important for UvrD mediated RecA displacement from ssDNA at damaged DNA replication forks.

Another interesting observation is that the UvrD monomer translocation rate is dependent on the ssDNA base composition where translocation was faster on pyrimidines than purines in the following order: dT>dC>dA>dG. The translocation rate decreased only 2-fold when changing from a ssDNA composed entirely of dT to a ssDNA composed of an equal mixture of each base. The ssDNA base specific effect on the translocation rate suggests the ssDNA base is an important element of the ssDNA recognized by UvrD during translocation, as suggested by the UvrD crystal structure. This is further supported by studies using abasic sites and polyethylene glycol spacers in the ssDNA where the absence of the ssDNA base has a greater effect on UvrD processivity than the absence of the ssDNA deoxyribose and phosphate moieties. Based on these results an inchworm stepping model for UvrD monomer translocation along ssDNA is proposed that is consistent with crystallographic studies of UvrD and PcrA; however, the presence of kinetic heterogeneity in the UvrD population adds an additional level of complexity to the mechanism, which cannot be assessed from static structures.

Language

English (en)

Chair and Committee

Timothy M. Lohman

Committee Members

Peter M. J. Burgers, Carl Frieden, Ellliot Elson, Enrico Di Cera, Nathan Baker

Comments

Permanent URL: https://doi.org/10.7936/K7C24TCQ

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