Control of DNA replication and its response to physiological and environmental changes is one of the fundamental regulatory challenges of living organisms. Understanding these interrelationships is important for understanding and interfering with normal and anomalous cell proliferation as well as with the parasitic relationships between viruses and their hosts. We are using phage T4 as a model system to study such interrelationships, because individual genes and gene products of this virus and its host, E. coli, are well characterized by in vivo and in vitro studies. Our overall goal is to understand control of DNA replication and its interrelationship with other DNA transactions (recombination, transcription, repair and packaging) in the context of development and of different environmental conditions. Our working hypothesis is that different replication and recombination modes reflect adaptations to other aspects of virus development and interactions with its host In this context, we want to study by combinations of genetic and biochemical approaches, the following problems and processes. 1) Origins of DNA replication that are used differentially under different growth conditions. Which signals and signal transductions are important for activating the different origins? How do structure and regulation of different transcripts and of certain DNA sequences in the origin regions affect primer synthesis, primer processing and assembly of replisomes? 2) The roles of different recombination pathways in recombination- dependent initiation of DNA replication and in DNA packaging. d) Do the different replication/recombination pathways differ in the fidelity of maintaining correct DNA sequence or gross overall chromosomal structure? 3) The roles of replication origins and of recombination in exclusion of one virus by another. 4) The relationship of prereplicative transcription, of palindromic putative transcription termination regions and overlapping late promoters in replication origin regions. Our main approach is to isolate DNA intermediates made in vivo after infection of host cells with different single and multiple virus mutants and to characterize and/or process these intermediates by biochemical and biophysical means and with purified proteins. Understanding the interconnections among regulatory circuits of living systems is important for understanding normal growth and development as well as disease. Specifically, the realization that topoisomerase deficiencies interfere with lagging strand DNA synthesis and as a consequence activate a specific recombination pathway is relevant to the use of topoisomerase inhibitors in the treatment of bacterial infections and of cancer cells and the generation of drug-resistant gene amplifications in treated cancer cells.
