E coli reca

The reaction contained 10 mM Tris-HCl pH7.5, 50 mM NaCl, 3 mM MgCl₂, 1.5 mM ATP, 1 mM DTT, 10 mM phosphocreatine, 35 U mL⁻¹ phosphocreatine kinase, 25 nM molecules (36 μM nt) of 5´ junction DNA with overhang labeled with Cy5. Rad51 at a final concentration of 12 μM (one protein per 3 nt) was introduced into the reaction and incubated 10 min followed by RPA at a final concentration of 1.44 μM (one protein per 25 nt) also during 10 min. Rad54 (wild-type Rad54 or Rad54K341R as stated in the results) and dsDNA (homologous or heterologous, as stated in the results) were added together in a final concentration of 175 nM and 25 nM in molecules of dsDNA (seven proteins per dsDNA molecule) in a final volume of 14 μL during 20 min. For the homologous donor, pUC19 plasmid was used, while PhiX174 RFI was used as heterologous DNA, both purchased from New England Biolabs and purified on MiniQ ion exchange chromatography column. This assay was carried out at 30 °C. In these reactions lacking Rad54 (control), storage buffer was added in order to rule out non-specific effects of buffer components and to maintain the ionic strength in all samples. For RecA D-loop assay, similar conditions were used, excepted the buffer was replaced by 30 mM of Tris-HCl and 9 mM of MgCl₂ and 1 mM of ATPγS with E. coli RecA (New England Biolabs) at 12 μM and E. coli SSB (New England Biolabs) protein at 0.6 μM.

Both wild-type and fluorescently labeled E. coli SSB were expressed and purified as described previously (Lohman et al., 1986 (link); Roy et al., 2009 (link)), with an addition of a dsDNA cellulose column to remove a minor exonuclease contaminant (Bujalowski and Lohman, 1991b (link)). The labeled SSB was single-point mutated from Ala to Cys at position 122 in the C-terminus, and labeled with AlexaFluor555 maleimide (Invitrogen, Grand Island, NY) to the extent of ∼25% (∼1 dye per tetramer) as described previously (Roy et al., 2009 (link)). E. coli RecA was purchased from New England Biolabs (M0249S; Ipswich, MA).

We incubated (37°C for 5 min) 0.5 μM each of (His)₆-tagged mouse RAD51, (His)₆-tagged mouse DMC1 or (His)₆-tagged E. coli RecA (New England Biolabs) with 5′-end ³²P-labeled 80-mer Oligo 1 ssDNA (1.5 μM nucleotides) to assemble filament in 8 μl reaction buffer C to which was added 2 mM MgCl₂ and 0.1 mM ATP. Then, indicated amounts of tag-free MCPH1 were included in the reaction mixtures for a further 5 min. To challenge filament stability, Benzonase (Endonuclease, 20 units; New England Biolabs) was added into the reaction mixtures (10 μl final volume). After 10 min of incubation at 37°C, the reaction mixtures were mixed with 2.5 μl of termination buffer (240 mM EDTA, 2% SDS and 3.2 mg/ml proteinase K) and incubated at 37°C for 10 min. The reaction mixtures were resolved in 10% polyacrylamide gels in TBE buffer at 4°C. The gels were dried on DE81 paper and the DNA species were quantified by phosphorimaging analysis in a Personal FX phosphorimager using the Quantity One software (Bio-Rad). The percentage of DNA protected was calculated as the ratio of the intensities of undigested DNA to total DNA, with the intensity of the DNA blank being defined as 100%.