Abstract
Escherichia coli UvrD protein is a 3' to 5' SF1 helicase required for DNA repair as well as DNA replication of certain plasmids. We have shown previously that UvrD can self-associate to form dimers and tetramers in the absence of DNA, but that a UvrD dimer is required to form an active helicase-DNA complex in vitro. Here we have used pre-steady state, chemical quenched flow methods to examine the kinetic mechanism for formation of the active, dimeric helicase-DNA complex. Experiments were designed to examine the steps leading to formation of the active complex, separate from the subsequent DNA unwinding steps. The results show that the active dimeric complex can form via two pathways. The first, faster path involves direct binding to the DNA substrate of a pre-assembled UvrD dimer (dimer path), whereas the second, slower path proceeds via sequential binding to the DNA substrate of two UvrD monomers (monomer path), which then assemble on the DNA to form the dimeric helicase. The rate-limiting step within the monomer pathway involves dimer assembly on the DNA. These results show that UvrD dimers that pre-assemble in the absence of DNA are intermediates along the pathway to formation of the functional dimeric UvrD helicase.
Highlights
DNA helicases are a ubiquitous class of enzymes that catalyze the separation of double-stranded[1] DNA to form the single-stranded DNA intermediates required for DNA replication, recombination, and repair in reactions that are coupled to the binding and hydrolysis of nucleoside triphosphates (1, 2)
The results of these studies show that the active, dimeric helicase can be formed either by rapid binding to the DNA substrate of pre-assembled UvrD dimers or by the sequential binding of two UvrD monomers followed by dimer assembly on the DNA
In previous studies (13) we have shown that a dimer of UvrD is required to observe helicase activity in single turnover DNA unwinding experiments in vitro
Summary
Buffers—Buffers were made with reagent grade chemicals using distilled water that was further deionized using a Milli-Q system (Millipore Corp., Bedford, MA). The second push rapidly mixed these reaction contents with Buffer T20 containing 1 mM ATP:Mg2ϩ, 2 M protein trap, and 1 M DNA trap This second mixing event initiates unwinding of any DNA on which an active dimeric UvrD helicase has assembled within the time, ⌬t1, while preventing any further binding of UvrD to the DNA substrate after the first incubation time (⌬t1). The other loop contained varying concentrations of the unlabeled 3Ј-(dT20)-ds[18] DNA substrate The contents of these two loops were rapidly mixed and allowed to incubate for ⌬t1 seconds, after which a second push mixed the solution with 1 mM ATP:Mg2ϩ to start the DNA unwinding reaction, along with 2 M protein trap and 1 M DNA trap, as described above. The UvrD species fraction distributions were calculated as described (17)
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