1. Volume 42, Issue 6, Pages 806-816 (June 2011)
Recombination Hotspots and Single-Stranded DNA Binding Proteins Couple DNA Translocation to DNA Unwinding by the AddAB Helicase-Nuclease 
Joseph T.P. Yeeles, Kara van Aelst, Mark S. Dillingham, Fernando Moreno-Herrero 
Molecular Cell 
Volume 42, Issue 6, Pages (June 2011)
DOI: /j.molcel
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2. Molecular Cell 2011 42, 806-816DOI: (10.1016/j.molcel.2011.04.012)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1
SSB Protein Stimulates DNA Unwinding by Suppressing Reannealing
(A) Schematic representation of the DNA substrates used in real-time helicase assays. B denotes biotin, S denotes streptavidin, and the red arrow indicates the position and orientation of the triple-Chi locus.
(B) STO helicase assays for the Chi0 (top) and Chi3 (bottom) substrates. Reactions were conducted in reaction buffer (see the Experimental Procedures) supplemented with 200 nM Hoechst and EcSSB (as indicated). AddAB (1 nM) was prebound to DNA (0.1 nM molecules, ∼750 nM ntds) that was blocked at the distal end with streptavidin (1 nM). Reactions were initiated by rapid mixing against an equal volume of reaction buffer supplemented with ATP (0.5 mM) and AddAHB mutant (30 nM).
(C) Final amplitudes of DNA unwinding (20 s time point) as a function of EcSSB (crosses) or BsSSB (open circles) concentration. Data for AddAB unwinding the Chi0 (black lines) and Chi3 (red lines) substrates, and for RecBCD unwinding the Chi0 substrate (blue squares), are shown.
(D) AddAB real-time triplex displacement assays with Chi0 DNA substrates performed in the absence of SSB. The triplex-forming oligonucleotide is located at different distances from the DNA end (as indicated). The lag times (x axis offsets) were plotted against the distance to the triplex, and the data was fit to a straight line to determine an AddAB translocation rate of 1800 bp s−1 (inset). Reactions were initiated by rapid mixing against an equal volume of reaction buffer supplemented with ATP (0.5 mM) and AddAHB trap (30 nM) for single-turnover conditions.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 DNA Reannealing Observed Directly by Atomic Force Microscopy
(A) Schematic representation of the Chi-free DNA substrate (Chi0AFM) used in atomic force microscopy.
(B) AFM image of reaction products involving AddAB, Chi0AFM DNA, and 1 mM ATP. This reaction yields three classes of product. Single-stranded DNA (yellow arrows, I), duplex DNA (black arrows, II), and mixed single-stranded and duplex DNA (green arrows, III) are shown. The interpretation of class I products as ssDNA was validated by comparing with images of heat-denatured DNA molecules (data not shown) and by labeling with SSB proteins.
(C) Products of reactions with a low concentration of ATP (10 μM ATP) were exclusively short duplex DNA molecules. AddAB appeared between gaps of fragmented dsDNA molecules (inset and black arrows) or bound at their ends (Figure S2A).
(D) Trapped intermediates of reactions involving the nuclease-deficient mutant AddANBN, Chi0AFM DNA, and 1 mM ATP. Examples of trapped AddAB complexes flanked by duplex DNA are highlighted by arrows and shown in the inset.
(E) Effect of BsSSB in Chi0AFM DNA reactions. Reactions included AddANBN, 1 mM ATP, and 1.5 μM BsSSB and produced filamentous SSB-ssDNA complexes (inset). All experiments shown in the figure were performed with 5 nM AddAB or AddANBN (where stated) and 0.3 nM DNA molecules incubated for 10 min at 37°C before adsorption.
The bar size in AFM images is 500 nm (main figures) and 250 nm (insets) and color scale from dark to bright is 0-2.5 nm (B–D) and 0–4 nm (E).
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Activation of DNA Unwinding Precisely at Chi Sequences
(A) Schematic representation of Chi3 DNA substrates with different pre-Chi distances.
(B) STO (continuous line) and MTO (dotted line) dye-displacement assays with Chi0 (black) and Chi3 (red) DNA substrates. Experiments were conducted with the conditions described in Figure 1 including 200 nM EcSSB, and 20 nM AddAHB trap for single turnover conditions.
(C) Detailed view of the first 1.5 s of the STO unwinding profile showing the activation of DNA unwinding induced by Chi recognition after a delay of approximately 400 ms. The dotted orange line shows the expected time it would take the enzyme to arrive at the Chi sequence based on triplex displacement data (see E below).
(D) STO dye-displacement assays were conducted with the different Chi3 DNA substrates (continuous line) and corresponding Chi0 DNA substrates (dotted line). Experiments were repeated with independent preparations of the DNA substrates and similar results were obtained. A representative data set is shown here. Chi3 DNA substrates display unwinding in two distinct phases; an early slower phase (comparable to the Chi0 substrates) followed by a later rapid phase.
(E) Time of unwinding activation as a function of the distance to Chi. Each activation time was obtained from the intercept between linear fits to the two unwinding phases for the Chi3 set of substrates.
In (E), data were fit to a straight line to determine an AddAB translocation rate of 1730 bp s−1 before Chi. The dotted pink line shows a fitted line to triplex displacement data (see Figure 1D), which provides an independent estimate of the DNA translocation rate of 1800 bp s−1. The line crosses the y axis at 80 ms. Based on these data, the predicted time for AddAB to reach Chi from the DNA end (for L = 525 bp) is shown by the vertical dotted line in (C).
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Chi-Dependent Activation of Unwinding via the Formation of ssDNA Loops
(A) Schematic representation of the Chi-containing DNA substrates used for AFM imaging. The arrows indicate the position and orientation of the triple-Chi loci.
(B) Atomic force microscopy image of reaction products involving AddANBN, Chi3AFM DNA, and 1 mM ATP. In addition to the three classes of products observed for Chi0AFM DNA, this reaction yielded a new type of product (class IV) that consisted of a ssDNA structure flanked by two tails of duplex DNA.
(C) Examples of class IV products for the Chi3AFM and Chi3AFM2 substrates. The lower row shows examples of class IV products of Chi3AFM in reactions supplemented with BsSSB (300 nM, left; 600 nM, middle; and 900 nM, right), which confirmed that the internal structure flanked by duplex DNA tails was indeed single-stranded DNA.
(D) Histogram showing the distribution of duplex tail lengths for class IV products of reactions involving the Chi3AFM and Chi3AFM2 substrates. The data were fit to a double-Gaussian function to yield an estimate of the tail lengths for each substrate.
(E) Two AddANBN enzymes from each DNA end collide at an intermediate point and produce a class IV structure with duplex DNA tails of lengths equal to the distance between Chi and the nearest DNA end.
All experiments shown in the figure were performed with 5 nM AddANBN and 0.3 nM DNA molecules incubated for 10 min at 37°C before adsorption. The scale bar size in AFM images is 500 nm (B) and 150 nm (C) and color scale from dark to bright is 0–2.5 nm (B and C).
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Model for the Enhancement of AddAB Helicase Activity by SSB and Chi Sequences
AddAB binds tightly to a break in a DNA molecule containing a Chi sequence (1). Upon ATP hydrolysis, AddAB advances along the DNA and unwinds the duplex to expose nascent single-strands of DNA for potential binding by SSB protein (2). At low or zero SSB concentrations, the nascent ssDNA rapidly reforms duplex (pathway i), whereas at high SSB concentrations the nascent ssDNA is stabilized by SSB (pathway ii) (3). Upon further ATP hydrolysis AddAB continues to translocate through the duplex and reannealing or unwinding continues (pathway i or ii) with occasional cleavage of the trailing DNA strands (4). Note that the reannealed duplexes are substrates for further processing by additional AddAB enzymes. Upon Chi-recognition and further ATP hydrolysis, AddAB continues to translocate but remains bound to Chi on the 3′-terminated strand (5). This generates a ssDNA loop downstream of Chi which both prevents degradation of the 3′-terminated strand and suppresses reannealing, thereby activating unwinding in pathway i.
Molecular Cell  , DOI: ( /j.molcel )
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