The main objective of the studies outlined in this proposal is to contribute to the understanding of DNA replication. Our immediate aim is to determine the cause of the abnormal reinitiation events that occur when phage gamma infects E. coli under SOS conditions. Normally, gamma replication is controlled such that daughter origins within replicative intermediates are inactive; only after termination of replication do they support initiation of further rounds of replication. Under SOS conditions, however, they are able to initiate during a round of replication; apparently the control of initiation is then profoundly disturbed. Details of normal and abnormal initiation events will be studied in vivo and in vitro. Effects of known mutations within the phage and host will be studied (+ SOS) by determination of replication ability and isolation of replicative intermediates. In vitro experiments will involve plasmid containing the gamma origin reacted with the initiator protein O (which produces an unwound DNA area within the origin if superhelical tension is present) along with various proteins connected with the SOS response. A comparison of the effects of HU and IHF protein on initiation in gamma and R6K will also be made. The studies will include S1 or P1 nuclease sensitivity, EM and gel electrophoresis analysis. These studies will yield information on DNA replication in more complex systems. In particular, a knowledge of how SOS conditions disturb the normal control of initiation of replication is of importance for the eventual understanding of normal and abnormal growth in higher organisms. Another major aim is to learn more about the recA protein mediated strand exchange reaction. This in vitro study, carried out in collaboration with Dr. Cox, will be concerned with the triple-strand pairing intermediate and also a newly discovered strand breakage reaction that occurs under certain conditions when strand exchange passes a heterogeneous barrier. This information should offer insights on the recombinational repair function that guards cells against the mutagenic or carcinogenic effect of DNA lesions. The final major aim is concerned with FLP protein promoted site-specific recombination. The intermediates in the reaction will be studied to gain insights into the isomerization of Holliday structures and how DNA sequence modulates the reaction. DNA-protein, DNA-DNA and protein-protein interaction will be studied. This information should ultimately be relevant to an understanding of recombination events during gene regulation.
