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. 2024 Jan 15;436(2):168381.
doi: 10.1016/j.jmb.2023.168381. Epub 2023 Dec 9.

E. coli RecB Nuclease Domain Regulates RecBCD Helicase Activity but not Single Stranded DNA Translocase Activity

Nicole T Fazio  1 , Kacey N Mersch  1 , Linxuan Hao  1 , Timothy M Lohman  2
Affiliations

E. coli RecB Nuclease Domain Regulates RecBCD Helicase Activity but not Single Stranded DNA Translocase Activity

Nicole T Fazio et al. J Mol Biol. .

Abstract

Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as occurring by the ATPase motors mechanically pulling the DNA duplex across a wedge domain in the helicase, biochemical data show that processive DNA unwinding by E. coli RecBCD helicase can occur in the absence of ssDNA translocation by the canonical RecB and RecD motors. Here we show that DNA unwinding is not a simple consequence of ssDNA translocation by the motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecBΔNucCD) unwinds dsDNA at significantly slower rates than RecBCD, while the ssDNA translocation rate is unaffected. This effect is primarily due to the absence of the nuclease domain since a nuclease-dead mutant (RecBD1080ACD), which retains the nuclease domain, showed no change in ssDNA translocation or dsDNA unwinding rates relative to RecBCD on short DNA substrates (≤60 base pairs). Hence, ssDNA translocation is not rate-limiting for DNA unwinding. RecBΔNucCD also initiates unwinding much slower than RecBCD from a blunt-ended DNA. RecBΔNucCD also unwinds DNA ∼two-fold slower than RecBCD on long DNA (∼20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecBD1080ACD unwinding are intermediate between RecBCD and RecBΔNucCD. Surprisingly, significant pauses in DNA unwinding occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, possibly allosterically and that RecBΔNucCD may mimic a post-chi state of RecBCD.

Keywords: DNA motor regulation; allostery; recombination; single molecule optical trap; transient kinetics.

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Conflict of interest statement

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1.
Figure 1.. Two classes of cryo-EM structures of RecBCD bound to blunt-ended DNA.
RecB motor domain (red), RecB nuclease domain (magenta), RecC (blue), RecD (green), and DNA (yellow). (A) RecBCD can melt up to 11 bp of DNA from a blunt end. Eleven bp are observed to be melted in one class of structures for which densities for the whole RecD subunit and RecBNuc are observed (PDB: 7MR3)[18]. (B) RecBCD melts only ~4 bp from a blunt end in a second class of structures for which the RecBNuc and RecD 2B domain densities are not observed (PDB: 7MR4)[18].
Figure 2.
Figure 2.. Representative stopped-flow time courses for RecBCD, RecBΔNucCD, and RecBD1080ACD unwinding from a blunt DNA end (buffer M30 at 37°C, 5 mM ATP).
(A) blunt-ended DNA substrate. (B) DNA unwinding traces monitoring the Cy3 fluorescence signal for RecBCD (red) and RecBΔNucCD (blue) (L = 50 bp). (C) DNA unwinding traces monitoring the Cy5 fluorescence signal for RecBCD (red) and RecBΔNucCD (blue) (L = 50 bp). Representative time courses for DNA unwinding of blunt ended DNA of L = 25 (black), 30 (red), 40 (blue), 50 (green), 60 (purple) bp by (D) RecBCD, (E) RecBΔNucCD, and (F) RecBD1080ACD. Global non-linear least square fits using Scheme 1 are shown as black solid lines. The best-fit parameters for the RecBCD data are: kU = 1306 s−1, m = 1.32 bp step−1, kNP = 12.3 s−1, kC = 151 s−1, h = 3.2 steps, x = 0.75, and mkU = 1726 bp s−1. The best-fit parameters for the RecBΔNucCD data are: kU = 225 s−1, m = 5.30 bp step−1, kNP = 1.0 s−1, kC = 4 s−1, h = 1.3 steps, x = 0.62, and mkU = 1351 bp s−1. The best-fit parameters for the RecBD1080ACD data are: kU = 643 s−1, m = 2.92 bp step−1, kNP = 4.67 s−1, kC = 106 s−1, h = 1.6 steps, x = 0.59, and mkU = 1879 bp s−1. (G) Average macroscopic unwinding rates from blunt-ended DNA for RecBCD, RecBΔNucCD and RecBD1080ACD.
Figure 3.
Figure 3.. Representative stopped-flow time courses for RecBCD, RecBΔNucCD, and RecBD1080ACD unwinding from a pre-melted 3’-dT6/5’-dT10 DNA end (buffer M30 at 37°C, 5 mM ATP).
(A) Pre-melted 3’-dT6/5’-dT10 DNA substrate. DNA unwinding traces monitoring the Cy3 fluorescence signal for RecBCD (red) and RecBΔNucCD (blue) (L = 50 bp). (C) DNA unwinding traces monitoring the Cy5 fluorescence signal for RecBCD (red) and RecBΔNucCD (blue) (L = 50 bp). Representative time courses for DNA unwinding of blunt ended DNA of L = 25 (black), 30 (red), 40 (blue), 50 (green), 60 (purple) bp by (D) RecBCD, (E) RecBΔNucCD, and (F) RecBD1080ACD. Global non-linear least square fits using Scheme 1 are shown as black solid lines. The best-fit parameters for the RecBCD data are: kU = 1105 s−1, m = 1.64 bp step−1, kNP = 11.3 s−1, kC = 108 s−1, h = 1.2 steps, x = 0.73, and mkU = 1813 bp s−1. The best-fit parameters for the RecBΔNucCD are: kU = 669 s−1, m = 1.83 bp step−1, kNP = 4.8 s−1, kC = 29 s−1, h = 1.1 steps, x = 0.44, and mkU = 1226 bp s−1. The best-fit parameters for the RecBD1080ACD are: kU = 897 s−1, m = 1.90 bp step−1, kNP = 8.10 s−1, kC = 139 s−1, h = 1.5 steps, x = 0.61, and mkU = 1701 bp s−1. (G) Average macroscopic unwinding rates from blunt-ended DNA for RecBCD, RecBΔNucCD and RecBD1080ACD.
Figure 4.
Figure 4.. Single molecule DNA unwinding by RecBCD, RecBΔNucCD and RecBD1080ACD.
(A) In a LUMICKS C-Trap, RecBCD and RecBCD variants are bound to a blunt-ended 19,435 bp DNA with the other end attached to a 4.34 μm streptavidin (SA) coated bead via a biotin-SA linkage in the presence of Sytox orange (Sxo) fluorophore that binds duplex DNA. In the presence of ATP, RecBCD starts to unwind the DNA, releasing the Sxo fluorophore. In the case of RecBCD, the newly formed single-stranded DNA is also degraded by its nuclease domain (magenta). (B) Representative plots showing the time dependence of the shortening of the duplex DNA lengths to obtain unwinding rates for RecBCD (1676 bp/s), RecBD1080ACD (1099 bp/s), and RecBΔNucCD (829 bp/s). These data show trajectories that do not contain significant pauses. (C) Rates of double-stranded DNA unwinding estimated only for trajectories that do not show pauses: RecBCD: 1612 ± 572 bp/s (n = 84 molecules), RecBD1080ACD: 1084 ± 503 bp/s (n = 26), and RecBΔNucCD: 723 ± 420 bp/s (n = 42) (mean ± SD (n)). Student’s T-test reveal that both RecBD1080ACD & RecBΔNucCD unwind DNA significantly slower than WT RecBCD (p-value <0.0001) and that RecBΔNucCD unwinds DNA significantly slower than RecBD1080ACD (p-value = 0.0022). (D), (E), and (F) Time-lapse of enzyme mediated shortening of DNA by (D) - RecBCD, (E) - RecBD1080ACD, and (F) - RecBΔNucCD.
Figure 5.
Figure 5.. RecBCD and RecBCD variants show chi-independent pausing during DNA unwinding.
Single DNA unwinding experiments performed on a 19,435 bp DNA with a LUMICKS C-Trap (A), (B) and (C) Examples of duplex DNA shortening trajectories displaying a single pause (shaded in gray) in DNA unwinding for (A) – RecBCD, (B) - RecBD1080ACD, and (C) - RecBΔNucCD. (D) The percentage of DNA unwinding trajectories for RecBCD (n = 144), RecBD1080ACD (n = 55), and RecBΔNucCD (n = 72) containing no pauses (blue), one pause (orange), and more than one pause (black). (E) DNA unwinding rates estimated from all data sets with and without pauses: RecBCD 1615 ± 721 bp/s (e= 218 unwinding events, n = 144 molecules), RecBD1080ACD 1117 ± 596 bp/s (e= 95, n = 55), and RecBΔNucCD 883 ± 658 bp/s (e= 109, n = 72 molecules) (mean ± SD bp/s.
Figure 6.
Figure 6.. Stopped-flow time courses monitoring 3’ to 5’ ssDNA translocation of RecBCD, RecBD1080ACD, and RecBΔNucCD (M275 at 37°C, 5 mM ATP).
(A) Pre-melted DNA substrate used to measure 3’ to 5’ ssDNA translocation. Representative DNA translocation time courses monitoring the enhancement of Cy3 fluorescence occurring when the helicase reaches the 5’ end of the ssDNA for (B) - RecBCD, (C) - RecBD1080ACD, and (D) - RecBΔNucCD. Time courses were obtained as a function of the 5’-ssDNA tail length (L = 30 nt (black); 50 nt (red); 70 nt (blue); 90 nt (green)). Lag times are plotted as a function of (L(nt)) below each data set and the macroscopic ssDNA translocation rate is estimated from the inverse of the slope of the best-fit line. (E) Average 3’ to 5’ ssDNA translocation rates for RecBCD, RecBΔNucCD, and RecBD1080ACD in buffer M275 (solid) and buffer M500 (striped) at 37°C and 5 mM ATP.
Figure 7.
Figure 7.. Stopped-flow time courses monitoring 5’ to 3’ ssDNA translocation of RecBCD, RecBD1080ACD, and RecBΔNucCD (M275 at 37°C, 5 mM ATP).
(A) Pre-melted DNA substrate used to measure 5’ to 3’ ssDNA translocation. Representative DNA translocation time courses monitoring the enhancement of Cy3 fluorescence occurring when the helicase reaches the 5’ end of the ssDNA for (B) - RecBCD, (C) - RecBD1080ACD, and (D) - RecBΔNucCD. Time courses were obtained as a function of the 3’-ssDNA tail length (L = 30 nt (black); 50 nt (red); 70 nt (blue); 90 nt (green)). Lag times are plotted as a function of (L(nt)) below each data set and the macroscopic ssDNA translocation rate is estimated from the inverse of the slope of the best-fit line. (E) Average 5’ to 3’ ssDNA translocation rates for RecBCD, RecBΔNucCD, and RecBD1080ACD in buffer M275 (solid) and buffer M500 (striped) at 37°C and 5 mM ATP.
Scheme 1
Scheme 1
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