Supplemental Data The RecA Binding Locus of RecBCD Is a General Domain for Recruitment of DNA Strand Exchange Proteins

Pyruvate kinase, lactate dehydrogenase, and phosphoenol pyruvate were purchased from Sigma. Restriction endonucleases, shrimp alkaline phosphatase (SAP), and T4 polynucleotide kinase were from New England Biolabs. Covalently closed circular χ +-3F3H dsDNA (a pBR322 derivative, containing tandem χ-sequences (Anderson et al., 1999)) was purified using a QIAGEN " Maxi kit ". M13mp7 replicative form was purified by cesium chloride gradient centrifugation. χ +-3F3H dsDNA, linearized with NdeI restriction endonuclease, and M13mp7 dsDNA, linearized with BglII enzyme, were dephosphorylated with SAP, and 5'-end labeled with T4 polynucleotide kinase and [γ-32 P]ATP. Hydroxyapatite (Bio-Gel HTP Gel) was from Bio-Rad; and Sepharose Fast-flow and HiLoad TM 16/60 Superdex TM 200 columns were from Amersham Pharmacia. The 6xHis RecB nuc was affinity purified as reported by (Zhang and Julin, 1999) with the following changes. Cells (BL21(DE3)) were disrupted by sonication in lysis buffer (50 mM TrisHCl (pH 7.5), 0.5 M NaCl, 50 mM imidazole, 0.5% triton X-100, and 1 mM PMSF). The cleared lysate was applied to a Sepharose Fast-flow column charged with 0.2 M nickel sulphate, and equilibrated with Ni-A buffer containing 50 mM TrisHCl (pH 7.5), 0.5 M NaCl, and 50 mM imidazole. Fractions that eluted with NiB buffer which contained 150 mM TrisHCl (pH 7.5), 0.5 M NaCl, and 300 mM imidazole, were dialyzed against HA-A buffer containing 20 mM potassium phosphate (pH 7.0), 1 mM EDTA, 0.1 mM DTT and 1 mM PMSF, and were applied to the hydroxyapatite column.