Joint molecule formation by coordinated action of RecA, RecF, RecO, RecR, RecQ, RecJ, and SSB proteins. (A) Reaction scheme: 3′-end-labeled linear dsDNA (“dsDNA” and homologous supercoiled DNA (“scDNA”) are used as substrates. DNA processing and homologous pairing produce joint molecules (“JM”) that are detected by agarose gel electrophoresis. (B) Reactions with either pUC1950 or pUC19 (as indicted) as the scDNA. The scDNA was 20 μM (nucleotides) to permit comparison with C. Due to the lower scDNA concentration, the product yield was 10% for pUC1950 and 8% for pUC19 at 60 min. (Left two panels) Ethidium bromide staining. (Right two panels) Radioisotopic imaging of 3′-end-labeled EcoRI-digested linear dsDNA. (C) Two-dimensional gel electrophoresis of reaction products. DNA was analyzed by native gel electrophoresis in the first dimension; one of two replicate lanes is shown at the top. The other gel slice was subjected to electrophoresis in a second dimension using denaturing conditions. The illustration shows the reaction products, which are indicated by arrows; the other spots are nonspecific background contaminants present at time 0. From right to left, the yield of joint molecule products is 6.8%, 0.5%, and 0.2%.

Homologous DNA pairing requires RecA, RecO, RecR, and RecJ proteins and is stimulated by RecF and SSB proteins. Proteins present are indicated at the top of the gel. The complete reaction is shown in three reaction time courses (reactions 1, 3, and 12) to permit direct comparison to the adjacent reactions (reactions 2, 4–11, and 13, respectively), which were performed at the same time. From left to right, the yield of joint molecules at 60 min for these reactions is 19%, 10%, 32%, 0%, 0%, 0%, 0%, 36%, 0%, 0%, 0%, 15%, and 3%.

RecQ helicase stimulates dsDNA resection by RecJ nuclease but disrupts joint molecules. (A) Joint molecule formation as a function of RecQ at various RecJ concentrations (indicated at top of the gel) was assayed at 60 min. (B) Quantification of joint molecule formation expressed as the amount of product relative to the amount of linear dsDNA, which is limiting. Error bars represent the standard error from multiple experiments.

DNA pairing in the RecAFORQJ reaction is concerted. (Left panel) The standard reaction conditions were used for the “Coupled” reaction. (Middle panel) In one uncoupled reaction (labeled “RecQJFOR → RecA + SSB”), the linear dsDNA was incubated with RecFOR, RecQ, RecJ, and SSB for 30 min, ethanol precipitated, and then suspended in reaction buffer at the same DNA concentration; a mixture of RecA and SSB was added, followed by the scDNA, and then incubated for the times indicated. In the right panel (“RecQJ → RecAFOR + SSB”), the linear dsDNA was incubated with RecQ, RecJ, and SSB for 30 min, ethanol precipitated, and then suspended; a mixture of RecFOR, RecA, and SSB was added, followed by the scDNA. The yield of joint molecules at 60 min is 20%, 0%, and 5%, respectively.

The need for RecF protein in joint molecule formation becomes apparent at lower concentrations of the RecOR proteins. Reactions used the indicated RecO and RecR concentrations, and were for 60 min. (A) The RecO concentration was varied from 0 to 100 nM in the presence or absence of RecF at 1 μM RecR. (B) Quantification of joint molecule formation in A expressed relative to the amount of linear dsDNA. (C) The RecR concentration was varied from 0 to 1000 nM in the presence or absence of RecF at 15 nM RecO. (D) Quantification of joint molecule formation in C expressed relative to the amount of linear dsDNA. Error bars represent the standard error.

RecA730 protein can partially bypass the need for RecF, RecO, and RecR proteins. Proteins present are indicated at the top of the gel. From left to right, the yield of joint molecules at 60 min is 19%, 0%, 4%, 0%, 2%, and 0%.

Model for dsDNA break repair by the proteins of the RecF pathway. The exonuclease activity of RecJ resects duplex DNA by degrading in the 5′ → 3′ direction, stimulated by RecQ helicase and SSB. The resultant ssDNA tail is bound by SSB. (Left) The RecFOR complex binds at the ssDNA–dsDNA junction on the resected DNA, and loads RecA onto the ssDNA. (Right) The RecOR complex can also mediate exchange of RecA for SSB bound to ssDNA at sites away from the junction. The RecA-ssDNA nucleoprotein filament invades homologous recipient duplex DNA. RecJ can contribute to stabilization of the D-loop by degrading the displaced ssDNA after nicking (not shown) (Corrette-Bennett and Lovett 1995), whereas RecQ helicase may disrupt recombination intermediates by unwinding from 3′ → 5′ direction. At this point (not shown), the joint molecule can be converted to a Holliday junction and resolved, or disrupted after DNA synthesis to anneal with the second end of a dsDNA break.