Kinetic Control of Mg2+-dependent Melting of Duplex DNA Ends by Escherichia coli RecBC

C. Jason Wong, Timothy M. Lohman

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

Escherichia coli RecBCD is a highly processive DNA helicase involved in double-strand break repair and recombination that possesses two helicase/translocase subunits with opposite translocation directionality (RecB (3′ to 5′) and RecD (5′ to 3′)). RecBCD has been shown to melt out ∼ 5-6 bp upon binding to a blunt-ended duplex DNA in a Mg2+-dependent, but ATP-independent reaction. Here, we examine the binding of E. coli RecBC helicase (minus RecD), also a processive helicase, to duplex DNA ends in the presence and in the absence of Mg2+ in order to determine if RecBC can also melt a duplex DNA end in the absence of ATP. Equilibrium binding of RecBC to DNA substrates with ends possessing pre-formed 3′ and/or 5′ single-stranded (ss)-(dT)n flanking regions (tails) (n ranging from zero to 20 nt) was examined by competition with a fluorescently labeled reference DNA and by isothermal titration calorimetry. The presence of Mg2+ enhances the affinity of RecBC for DNA ends possessing 3′ or 5′-(dT)n ssDNA tails with n < 6 nt, with the relative enhancement decreasing as n increases from zero to six nt. No effect of Mg2+ was observed for either the binding constant or the enthalpy of binding (ΔHobs) for RecBC binding to DNA with ssDNA tail lengths, n ≥ 6 nucleotides. Upon RecBC binding to a blunt duplex DNA end in the presence of Mg2+, at least 4 bp at the duplex end become accessible to KMnO4 attack, consistent with melting of the duplex end. Since Mg2+ has no effect on the affinity or binding enthalpy of RecBC for a DNA end that is fully pre-melted, this suggests that the role of Mg2+ is to overcome a kinetic barrier to melting of the DNA by RecBC and presumably also by RecBCD. These data also provide an accurate estimate (ΔHobs = 8 ± 1 kcal/mol) for the average enthalpy change associated with the melting of a DNA base-pair by RecBC.

Original languageEnglish
Pages (from-to)761-777
Number of pages17
JournalJournal of Molecular Biology
Volume378
Issue number4
DOIs
StatePublished - May 9 2008

Keywords

  • fluorescence
  • helicase
  • kinetics and thermodynamics
  • motor protein
  • recombination

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