Influence of DNA End Structure on the Mechanism of Initiation of DNA Unwinding by the Escherichia coli RecBCD and RecBC Helicases

Abstract

E. 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” ~ six bp upon binding to the end of a blunt-ended DNA duplex in a Mg2+-dependent but ATP-independent reaction, we have investigated the effects of non-complementary 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 non-complementary 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 non-complementary 5'-ssDNA tail is extended to ten nucleotides (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 non-complementary twin 5'- dT6 and 3'-dT6 tails, and unwinding can be described by a simple uniform n-step sequential scheme, without the need of 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 (dT6)–tailed DNA reflect processes needed to engage the RecD motor with the 5' ssDNA.

RecA4142 Causes SOS Constitutive Expression by Loading onto Reversed Replication Forks inEscherichia coliK-12

“…RecBCD loads preferentially onto fairly blunt DSEs of DNA (43,45). Since the DSE produced during RFR by the (20).…”

Asymmetric Regulation of Bipolar Single-stranded DNA Translocation by the Two Motors within Escherichia coli RecBCD Helicase

“…Heparin stock solutions were stored at 4°C until use, and its concentration was determined by titration with Azure A as described (21). ATP stock solutions were prepared and stored at Ϫ20°C as described (22).…”

“…RecBC was reconstituted by mixing RecB and RecC at equimolar concentrations on ice. RecBC was shown to fully form heterodimers by sedimentation velocity experiments in buffer M. The enzymes were dialyzed against buffer M 30 at 4°C before use, and enzyme concentrations were determined spectrophotometrically using an extinction coefficient of ⑀ 280 ϭ 3.9 ϫ 10 Oligodeoxynucleotides-Oligodeoxynucleotides, either unlabeled or labeled covalently with Cy3 or fluorescein, were synthesized and purified, and their concentrations were determined as described (22). DNA stocks were stored in buffer M 30 in the absence of Mg 2ϩ at Ϫ20°C until use.…”

“…DNA substrates with various duplex lengths possessing a high affinity binding site (a fork with a 3Ј-(dT) 6 and a 5Ј-(dT) 10 tail) were described previously (22). Final concentrations after mixing were 150 nM WT and mutant RecBCD, 50 nM DNA, and 7.5 mg/ml heparin in buffer M 250 plus 10 mM MgCl 2 and 5 mM ATP.…”

Kinetics of DNA Unwinding by the RecD2 Helicase from Deinococcus radiodurans

“…Unwinding rates for other SF1 helicases that are more distantly related to RecD2 vary from 30 bp/s for PcrA (32)) and 68 bp/s for UvrD (41)), to 790 bp/s for RecBCD (28). However, the kinetic step sizes of these enzymes are similar to that of RecD2: UvrD (4 -5 bp/step (27)), PcrA (4 bp/step (35)), and RecBC and RecBCD (ϳ3-4 bp/step (28,37)). The similarity in kinetic step size suggests an underlying mechanistic similarity in DNA unwinding by these helicases, despite the differences in their unwinding rates.…”