Fig. 1.

Reconstitution of recombination-associated DNA synthesis. (a) A schematic representation of the reaction. First, RAD51 (1 μM) was loaded onto a 90 nt D1 oligonucleotide (3 μM as nucleotides) for 5 min at 37 °C. Hop2/Mnd1 complex (200 nM) was then incorporated and the mixture was incubated for 1 min at 37 °C. The reaction was started by adding pBluescript SK(−) replicative form I (50 μM as base pairs) and incubating for 5 min at 37 °C. Next, PCNA (30 nM) was loaded by RFC complex (10 nM) in the presence of RPA (666 nM) and dNTPs (100 μM each) for 5 min at 30 °C. The extension was initiated by incorporation of Pol δ (30 nM). After 10 min at 37 °C, reactions were stopped, treated with proteinase K, and then analyzed. (b) All tested proteins are required for efficient DNA repair synthesis. The reactions were performed as described above. Individual proteins were omitted as indicated. α-[³²P]-dATP was used to follow the reactions. Labeled lambda DNA digested with BstEI was used as a marker (only a subset of bands is depicted).