DNA synthesis by two eukaryotic DNA polymerases, α and δ, was studied using a single-strand M13 DNA template primed at a unique site. In the presence of low amounts of either DNA polymerase α or δ, DNA synthesis was limited and short DNA strands of ~ 100 bases were produced. Addition of replication factors RF-A, PCNA and RF-C, which were previously shown to be required for SV40 DNA replication in vitro, differentially stimulated the activity of both DNA polymerases. RF-A and RF-C independently stimulated DNA polymerase α activity 4- to 6-fold, yielding relatively short DNA strands (< 1 kb) and PCNA had no effect. In contrast, polymerase δ activity was stimulated co-operatively by PCNA, RF-A and RF-C ~ 25- to 30-fold, yielding relatively long DNA strands (up to 4 kb). Neither RF-C nor RF-A appear to correspond to known polymerase stimulatory factors. RF-A was previously shown to be required for initiation of DNA replication at the SV40 origin. Results presented here suggest that it also functions during elongation. The differential effects of these three replication factors on DNA polymerases α and δ is consistent with the model that the polymerases function at the replication fork on the lagging and leading strand templates respectively. We further suggest that co-ordinated synthesis of these strands requires dynamic protein-protein interactions between these replication factors and the two DNA polymerases.
|Number of pages||7|
|Publication status||Published - 1989|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Immunology and Microbiology(all)