Kinetic control of Mg2+-dependent melting of duplex DNA ends by Escherichia coli RecBC.
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 approximately 5-6 bp upon binding to a blunt-ended duplex DNA in a Mg(2+)-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 Mg(2+) 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 Mg(2+) 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 Mg(2+) 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>or=6 nucleotides. Upon RecBC binding to a blunt duplex DNA end in the presence of Mg(2+), at least 4 bp at the duplex end become accessible to KMnO(4) attack, consistent with melting of the duplex end. Since Mg(2+) 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 Mg(2+) 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.
Wong
C Jason
CJ
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8231, Saint Louis, MO 63110-1093, USA.
Lohman
Timothy M
TM
D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D043211:Exodeoxyribonuclease V / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D011485:Protein Binding; D013379:Substrate Specificity; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D004247:DNA; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D008274:Magnesium