The RecAQ-coupled pairing reaction produces two classes of joint molecules. Each lane represents a 10 min time point taken from the indicated reaction. (Lanes 1–3) scDNA recipient in each reaction was different to determine the position of the band corresponding to scDNA-dependent D-loops. (Lane 1) Taken from a standard coupled reaction, with the deduced migration positions of each joint molecule indicated to the left. The products shown in lanes 2 and 3 are from reactions with larger, homologous pUCD scDNA and nonhomologous φX174 RF I, respectively. The migration of the D-loop band is slower for the larger pUCD but the same band is completely absent when φX174 RF I is used in its place. (Lanes 4,5) scDNA was omitted from reactions to identify scDNA-independent Kappa intermediates. (Lane 4) Band corresponding to the Kappa intermediate is absent at the prevailing RecQ helicase concentration (100 nm); however, the band is present with fivefold less RecQ helicase (lane 5), confirming that it is scDNA-independent.

 Model for RecQ helicase action in homologous recombination. (A) RecQ helicase initially bound to the 5′-end labeled linear dsDNA substrate unwinds these molecules to produce linear ssDNA. During the unwinding process, SSB protein binds to the liberated ssDNA, trapping it and, thereby, preventing the strands from reannealing. (B) This SSB protein-coated ssDNA is then bound by RecA protein to form a nucleoprotein filament competent for homologous pairing. The RecA nucleoprotein filament initiates homologous pairing and strand invasion to form joint molecules. The major species of joint molecules formed in these reactions are scDNA-dependent D-loops and linear scDNA-independent Kappa intermediates. (C) After the initial unwinding of linear dsDNA, RecQ helicase is available to bind to and dissociate the accumulated joint molecules.

 RecQ helicase, in conjunction with RecA and SSB proteins, initiates homologous pairing. Labeled, linear pUC19 DNA and pUC1950 scDNA were incubated with: (Lane 1) RecA and SSB proteins; (lane 2) RecQ helicase and SSB protein; (lanes 3–8) all three proteins; (lane 9) RecQ helicase and RecA protein alone. Slower migrating species that represent joint molecules, labeled Joint Molecules, are observed only in the presence of all three proteins. In a control reaction, RecA and SSB proteins were incubated with labeled linear pUC19 ssDNA, cold linear pUC19 dsDNA, and pUC1950 scDNA to produce bona fide joint molecules (lane 10). All reactions were carried out as in Materials and Methods. Bands corresponding to linear ssDNA and dsDNA are also indicated to the left.

 RecQ helicase dissociates joint molecules. (A) RecQ helicase unwinds joint molecules during the course of the coupled pairing reaction. The addition of fivefold more RecQ protein (500 nm) to the coupled pairing reaction results in transient intermediates and a lower steady-state yield of these species (lanes 2–5). The helicase activity of RecQ helicase is required to observe this phenomenon because addition of ATPγS, a nonhydrolyzable analog of ATP, affords protection from disruption to the pairing intermediates formed in the reaction (lanes 6–9). The time at which 4 mm ATPγS was added (8 min) is indicated by the vertical arrow. (B) RecQ helicase binds to and unwinds joint molecules formed in its absence. (Lane 1) Linear pUC19 dsDNA marker. (Lanes 2–6) RecA and SSB proteins were incubated with labeled linear pUC19 ssDNA, unlabeled linear pUC19 dsDNA, and pUC1950 scDNA to produce homologous pairing intermediates. RecQ helicase added to similar reactions dissociated the joint molecules formed by RecA protein (lanes 7–11). RecQ helicase (500 nm) was added after taking the 10 min time point in lane 9.