Figure 4.

RecA-mediated HR in the presence of heterologous ends. (A) Scheme of the lds substrate with heterology at the 3'- and 5'-end (in red) with the css (+) substrate and the expected product (prd). (B) Circular 3199 nt ssDNA (10 μM) and 4374 bp NcoI-linearized dsDNA substrate (20 μM in nt) were pre-incubated with SsbA (0.3 μM) for 5 min at 37°C in buffer A containing 5 mM dATP, ATP or ATPγS, then the preformed SsbA·ssDNA complex was incubated or not with RecO (0.2 μM) or DprA (0.3 μM) for 5 min at 37°C. Finally RecA (1.2 μM) was added, and the reaction was incubated for 30 or 60 min at 37ºC and products separated by 0.8% AGE. In lane 17, the cds DNA substrate was treated with DNase I to generate nc DNA. The positions of the bands corresponding to css, lds, prd and jm are indicated. The symbols + and – denote the presence or absence of the indicated protein(s). Amounts of recombination intermediates and products are indicated, and expressed as percentage of total substrate added. The results are the average of more than three independent experiments (the results given are within a 5% standard error).