A MAMMALIAN PROTEIN COMPLEX THAT REPAIRS DOUBLE-STRAND BREAKS AND DELETIONS BY RECOMBINATION

We have purified a high molecular weight complex (RC-1) from calf thym us nuclei that catalyzes a recombinational repair of double-strand gap s and deletions in DNA by gene conversion as well as cross-over events leading to cointegrant products. These have been detected by polymera se chain reaction analysis using oligonucleotide primer pairs that det ect joined sequences originally present on only one or the other of th e recombination substrates. RC-1 has an apparent molecular mass of abo ut 550-600 kDa and contains at least five polypeptide chains: molecula r masses about 230, 210, 160, 130, and 40 kDa. RC-1 contains a DNA pol ymerase, identified as DNA polymerase epsilon, that copurifies with RC -1. A DNA ligase, most likely mammalian DNA ligase III, and a 5'-3' ex onuclease also copurify with the RC-1. Most preparations of RC-1 conta in low levels of a double-strand endonuclease, 3'-5' exonuclease and s ingle-strand nuclease activities. However, DNA helicase, terminal deox ynucleotidyl transferase, or DNA topoisomerase I and II were not detec ted in RC-1. The DNA polymerase and DNA ligase in RC-1 can act in conc ert to repair a multiply gapped DNA to a covalently repaired duplex. T he bovine single-strand-binding protein stimulates the formation of th e recombination products and the repair reaction mentioned above about 4-fold.