The aim of this project is to understand more fully the process of DNA replication through detailed study of the mechanisms of selected DNA polymerases. The enzymes to be examined include: the prokaryotic DNA polymerases, E. coli DNA polymerases II and III; and the viral DNA polymerase, avian myeloblastosis virus (AMV) polymerase. Experiments will concentrate on measurement of basic parameters of enzyme mechanism in vitro, specifically including processivity, i.e. the number of nucleotides added each time the polymerase binds DNA. We have recently developed a quantitative assay for measurement of processivity of DNA synthesis in vitro (Bambara et al., 1978). The availability of this new assay provides an opportunity to examine factors which perturb the binding and translocation mechanism of DNA polymerases, namely DNA replication-associated proteins and drugs which inhibit DNA synthesis. Experiments will include measurement of the effects of: a. E. coli DNA binding protein on binding and processivity of E. coli DNA polymerase III. The processivity assay will be used to detect DNA replication proteins in extracts of chicken myeloblasts which affect the translocation mechanism of AMV polymerase. Later in the grant period, the same technique will be used to begin a search in calf thymus cell extracts for similar proteins which may increase the extent of processive DNA synthesis of calf thymus DNA polymerases. Inhibition of the AMV DNA polymerase by the intercalating antitumor drugs adriamycin and 9-hydroxyellipticine will be studied, with attention to direct effects of these drugs on the translocation mechanism of the DNA polymerase.