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. 2013 Jan 11;288(2):1055-64.
doi: 10.1074/jbc.M112.423384. Epub 2012 Nov 27.

Asymmetric regulation of bipolar single-stranded DNA translocation by the two motors within Escherichia coli RecBCD helicase

Fuqian Xie  1 , Colin G Wu, Elizabeth Weiland, Timothy M Lohman
Affiliations

Asymmetric regulation of bipolar single-stranded DNA translocation by the two motors within Escherichia coli RecBCD helicase

Fuqian Xie et al. J Biol Chem. .

Abstract

Repair of double-stranded DNA breaks in Escherichia coli is initiated by the RecBCD helicase that possesses two superfamily-1 motors, RecB (3' to 5' translocase) and RecD (5' to 3' translocase), that operate on the complementary DNA strands to unwind duplex DNA. However, it is not known whether the RecB and RecD motors act independently or are functionally coupled. Here we show by directly monitoring ATP-driven single-stranded DNA translocation of RecBCD that the 5' to 3' rate is always faster than the 3' to 5' rate on DNA without a crossover hotspot instigator site and that the translocation rates are coupled asymmetrically. That is, RecB regulates both 3' to 5' and 5' to 3' translocation, whereas RecD only regulates 5' to 3' translocation. We show that the recently identified RecBC secondary translocase activity functions within RecBCD and that this contributes to the coupling. This coupling has implications for how RecBCD activity is regulated after it recognizes a crossover hotspot instigator sequence during DNA unwinding.

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Figures

FIGURE 1.
FIGURE 1.
Fluorescence assay to monitor RecBCD translocation in the 3′ to 5′ and 5′ to 3′ directions. A, schematic of RecBCD bound to a DNA end. RecB motor (red), RecC (blue), and RecD motor (green), RecB nuclease (Nuc) domain (purple), and RecB arm region (orange). B, stopped-flow time course (red) monitoring RecBCD translocation along ssDNA in the 3′ to 5′ direction using DNA III. RecBCD binds at the loading site, upon addition of ATP and heparin (trap for free RecBCD) unwinds the 24-bp duplex, and then translocates along the (dT)L extension until it reaches the Cy3 fluorophore (red circle), resulting in Cy3 fluorescence enhancement. Dissociation of RecBCD returns the Cy3 fluorescence to the free DNA level (blue). AU, arbitrary units.
FIGURE 2.
FIGURE 2.
The rate of RecBCD translocation along ssDNA in the 5′ to 3′ direction is faster than in the 3′ to 5′ direction, and the rates are coupled asymmetrically. Stopped-flow experiments were performed by mixing a preformed complex of RecBCD (37.5 nm) and DNA (50 nm) with 5 mm ATP, 10 mm MgCl2, and 7.5 mg/ml heparin (concentrations after mixing) in buffer M250 at 25 °C. The lengths of the twin ssDNA extensions, L, are indicated. All insets show the linear dependence of lag time on ssDNA extension length L. A, monitoring 3′ to 5′ ssDNA translocation of RecBCD using DNA I. Inset, rate = 1409 ± 109 nt/s. B, monitoring 5′ to 3′ ssDNA translocation of RecBCD using DNA II. Inset, rate = 1922 ± 72 nt/s. C, monitoring 3′ to 5′ ssDNA translocation of RecBCDK177Q using DNA I. Inset, rate = 1289 ± 14 nt/s. D, monitoring 5′ to 3′ ssDNA translocation of RecBK29QCD using DNA II. Inset, rate = 1162 ± 11 nt/s. E, [ATP] dependence of the 3′ to 5′ translocation rates of RecBCDK177Q (red) and WT RecBCD (black). For WT RecBCD, Km = 337 ± 30 μm and Vmax = 1515 ± 33 nt/s; for RecBCDK177Q, Km = 327 ± 14 μm and Vmax = 1377 ± 14 nt/s. F, [ATP] dependence of the 5′ to 3′ translocation rates of RecBK29QCD (green) and WT RecBCD (black). For WT RecBCD, Km = 420 ± 89 μm and Vmax = 2037 ± 109 nt/s; for RecBK29QCD, Km = 534 ± 84 μm and Vmax = 1325 ± 56 nt/s. AU, arbitrary units.
FIGURE 3.
FIGURE 3.
Slowing the RecB motor (RecBY803H) slows both the 3′ to 5′ and 5′ to 3′ translocation rates, whereas slowing the RecD motor (RecDY567H) slows only the 5′ to 3′ translocation rate. A, monitoring 3′ to 5′ ssDNA translocation of RecBY803HCD using DNA I. Inset, rate = 441 ± 13 nt/s. B, monitoring 5′ to 3′ ssDNA translocation of RecBY803HCD using DNA II. Inset, rate = 1454 ± 75 nt/s. C, monitoring 3′ to 5′ ssDNA translocation of RecBCDY567H using DNA I. Inset, rate = 1412 ± 12 nt/s. D, monitoring 5′ to 3′ ssDNA translocation of RecBCDY567H using DNA II. Inset, rate = 1046 ± 108 nt/s. AU, arbitrary units.
FIGURE 4.
FIGURE 4.
The secondary translocase activity of RecBC is functional within RecBCDK177Q and WT RecBCD. A, time courses monitoring the secondary RecBC (5′ to 3′) ssDNA translocation of RecBCDK177Q using DNA substrate IV. Inset, rate = 1393 ± 10 nt/s. B, time course monitoring the secondary RecBC translocase within WT RecBCD (red trace) on DNA substrate V. The Cy3-labeled ssDNA extension (dT)45 contains a reverse polarity (3′ to 3′) linkage (red X). No translocation is observed for RecBK29QCD (blue trace) on the same DNA. AU, arbitrary units.
FIGURE 5.
FIGURE 5.
Comparison between ssDNA translocase activities and helicase activity within RecBCD. A, 3′ to 5′ translocase activity is correlated with the helicase activity. The linear fit is introduced to show the correlation. B, 5′ to 3′ translocase activity is not correlated with the helicase activity. Error bars represent S.D.
FIGURE 6.
FIGURE 6.
Schematic of RecBCD-DNA complexes pre- and post-CHI. A, RecBCD-DNA initiation complex. RecD (green) and the secondary RecBC translocation site (blue) operate on the 5′-ssDNA tail, and the primary RecBC translocase site (red) operates on the 3′-ssDNA tail. B, pre-CHI DNA unwinding by RecBCD. The 5′ to 3′ rate is faster due to the action of two translocases (RecD (green) and the secondary RecBC (blue)) versus only one translocase (primary RecBC) acting in the 3′ to 5′ direction, resulting in a loop in the 3′-terminated strand ahead of the enzyme. C, post-CHI DNA unwinding by RecBCD. Upon deactivation of RecD, the 3′ to 5′ and 5′ to 3′ translocation rates are equal due to the concerted action of the RecBC secondary (5′ to 3′) (blue) and primary (3′ to 5′) (red) translocases, resulting in maintenance of the loop formed in the 3′-terminated strand ahead of the enzyme. Two alternative pathways (I versus II) result depending on whether CHI remains bound to RecC (I) wherein a second loop would form at the interface between RecB and RecC as proposed (13, 41). Alternatively, if the 3′-tail does not remain bound to RecC (II), then the 3′-ssDNA will pass through RecC, and a second loop would not form.
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