DNA-SYNTHESIS ON DISCONTINUOUS TEMPLATES BY HUMAN DNA-POLYMERASES - IMPLICATIONS FOR NONHOMOLOGOUS DNA RECOMBINATION

DNA polymerases catalyze the synthesis of DNA using a continuous unint errupted template strand, However, it has been shown that a 3'-->5' ex onuclease-deficient form of the Klenow fragment of Escherichia coil DN A polymerase I as well as DNA polymerase of Thermus aquaticus can synt hesize DNA across two unlinked DNA templates. In this study, we used a n oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well as fractionation of the extracts revealed that most of the disco ntinuous DNA synthesis was attributable to DNA polymerase alpha. Addit ionally, discontinuous DNA synthesis could be eliminated by incubation with an antibody that specifically neutralized DNA polymerase alpha a ctivity. To test the relative efficiency of each nuclear DNA polymeras e for discontinuous synthesis, equal amounts (as measured by DNA polym erase activity) of DNA polymerases alpha, beta, delta (+/- PCNA) and e psilon (+/- PCNA) were used in the discontinuous DNA synthesis assay, DNA polymerase alpha showed the most discontinuous DNA synthesis activ ity, although small but detectable levels were seen for DNA polymerase s delta (+PCNA) and epsilon (PCNA), Klenow fragment and DNA polymerase beta showed no discontinuous DNA synthesis, although at much higher a mounts of each enzyme, discontinuous synthesis was seen for both, Disc ontinuous DNA synthesis by DNA polymerase alpha was seen with substrat es containing 3 and 4 bp single-strand stretches of complementarity; h owever, little synthesis was seen with blunt substrates or with 1 bp s tretches, The products formed from these experiments are structurally similar to that seen in vivo for non-homologous end joining in eukaryo tic cells. These data suggest that DNA polymerase alpha may be able to rejoin double-strand breaks in vivo during replication.