This project is a further attempt to elucidate the mechanism of DNA replication in E. coli. Several specific approaches will be taken to various aspects of this overall problem. 1) An investigation into the nature of the 4S DNA intermediate observed in dnaB mutants both in vivo and in vitro and previously reported. Both the relationship of 4S intermediates to 10S intermediates and the genesis of the 4S DNA will be studied. 2) The isolation, genetic mapping, and physiological classification of suppressors of various dna alleles will be used to construct an interaction map of the DNA synthesis machinery and is expected to provide new mutations involved primarily or secondarily with DNA replication. Two such suppressors which appear to affect membrane structure will be further characterized. 3) A dnaE mutant which exhibits arithmetic synthesis at high temperature has been shown to respond abnormally to antibiotics which affect initiation of replication. The effects of this mutation make it likely that DNA synthesis can be examined as if it were composed of two separate controlling elements.