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” ∼ 6 bp upon binding to the end of a blunt-ended DNA duplex in a Mg2+-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 (mkU = 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 (kC = 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 (mkU = 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 kC initiation steps, with a similar kinetic step size (m = 4.4 ± 1.7 bp) and unwinding rate (mkobs = 396 ± 15 bp s− 1). These results suggest that the additional kinetic steps with rate constant kC 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.