Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases.
Escherichia coli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3'-to-5' translocase, and RecD, a 5'-to-3' translocase) that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can "melt out" approximately 6 bp upon binding to the end of a blunt-ended DNA duplex in a Mg(2+)-dependent but ATP-independent reaction, we investigated the effects of noncomplementary single-stranded (ss) DNA tails [3'-(dT)(6) and 5'-(dT)(6) or 5'-(dT)(10)] on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3'-(dT)(6) and 5'-(dT)(6) noncomplementary ssDNA tails is well described by a sequential n-step mechanism with the same unwinding rate (mk(U)=774+/-16 bp s(-1)) and kinetic step size (m=3.3+/-1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding (k(C)=45+/-2 s(-1)). However, when the noncomplementary 5' ssDNA tail is extended to 10 nt [5'-(dT)(10) and 3'-(dT)(6)], the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate (mk(U)=538+/-24 bp s(-1)) is observed with a similar kinetic step size (m=3.9+/-0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing noncomplementary twin 5'-(dT)(6) and 3'-(dT)(6) tails, and unwinding can be described by a simple uniform n-step sequential scheme, without the need for the additional k(C) initiation steps, with a similar kinetic step size (m=4.4+/-1.7 bp) and unwinding rate (mk(obs)=396+/-15 bp s(-1)). These results suggest that the additional kinetic steps with rate constant k(C) required for RecBCD to initiate unwinding of blunt-ended and twin (dT)(6)-tailed DNA reflect processes needed to engage the RecD motor with the 5' ssDNA.
Wu
Colin G
CG
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, Box 8231, St. Louis, MO 63110, USA.
Lohman
Timothy M
TM
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D043211:Exodeoxyribonuclease V / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism
D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D004247:DNA; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli