Kinetic control of Mg2+-dependent melting of duplex DNA ends by Escherichia coli RecBC

JOURNAL OF MOLECULAR BIOLOGY

卷:378

Article

Wong, C. Jason¹  Lohman, Timothy M.¹ 

[1] Washington Univ, Sch Med, Dept Biochem & Mol Biophys, St Louis, MO 63110 USA

关键词: fluorescence;    motor protein;    helicase;    recombination;    kinetics and thermodynamics;   

DOI  :  10.1016/j.jmb.2008.03.023

来源: Elsevier

【 摘 要 】

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 similar to 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 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 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 (Delta H-obs) 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 (Delta H-obs,=8 +/- 1 kcal/mol) for the average enthalpy change associated with the melting of a DNA base-pair by RecBC. (c) 2008 Elsevier Ltd. All rights reserved.

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