A dimer of Escherichia coli UvrD is the active form of the helicase in vitro

Nasib K. Maluf, Christopher J. Fischer, Timothy M. Lohman

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162 Scopus citations

Abstract

The Escherichia coli UvrD protein is a 3′ to 5′ SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. We have characterized in vitro UvrD-catalyzed unwinding of a series of 18bp duplex DNA substrates with 3′ single-stranded DNA (ssDNA) tails ranging in length from two to 40nt. Single turnover DNA-unwinding experiments were performed using chemical quenched flow methods, as a function of both [UvrD] and [DNA] under conditions such that UvrD-DNA binding is stoichiometric. Although a single UvrD monomer binds tightly to the single-stranded/double-stranded DNA (dsDNA) junction if the 3′ ssDNA tail is at least four nt, no unwinding was observed for DNA substrates with tail-lengths ≤8nt, even at high [UvrD]/[DNA] ratios. Unwinding is observed for DNA substrates with 3′ ssDNA tail lengths ≥12nt, and the unwinding amplitude displays a sigmoidal dependence on [UvrDtot]/[DNAtot]. Quantitative analysis of these data indicates that a single UvrD monomer bound at the ssDNA/dsDNA junction of any DNA substrate, independent of 3′ ssDNA tail length, is not competent to fully unwind even a short 18bp duplex DNA, and that two UvrD monomers must bind the DNA substrate in order to form a complex that is able to unwind short DNA substrates in vitro. Other proteins, including a mutant UvrD with no ATPase activity as well as a monomer of the structurally homologous E.coli Rep helicase, cannot substitute for the second UvrD monomer, suggesting a specific interaction between two UvrD monomers and that both must be able to hydrolyze ATP. Initiation of DNA unwinding in vitro appears to require a dimeric UvrD complex in which one subunit is bound to the ssDNA/dsDNA junction, while the second subunit is bound to the 3′ ssDNA tail.

Original languageEnglish
Pages (from-to)913-935
Number of pages23
JournalJournal of Molecular Biology
Volume325
Issue number5
DOIs
StatePublished - Jan 31 2003

Keywords

  • DNA unwinding
  • Helicase II
  • Mechanism
  • Repair
  • Single turnover kinetics

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