Mutational analysis of primosome assembly sites. I. Distinct classes of mutants in the pBR322 Escherichia coli factor Y DNA effector sequences

Abstract

The assembly of the primosome, a multienzyme complex responsible for priming of lagging-strand DNA synthesis in Escherichia coli, occurs on defined regions of DNA. These primosome assembly sites are on the order of 70 nucleotides in length, yet they share little DNA sequence homology. In order to understand the interaction of the primosomal proteins with these sites, the isolation of single-base substitution mutants of the wild-type sequences has been undertaken. The response of 32 of these mutated primosome assembly sites to increasing concentrations of monovalent and divalent cations when they were used as DNA effectors for E. coli replication factor Y-catalyzed ATP hydrolysis and their efficiency as primosome-dependent DNA replication templates have revealed the existence of four distinct classes of mutations in primosome assembly sites. Class I mutations have essentially no effect on the activities elicited by the DNA site; thus, it is likely that they define nonessential or spacer nucleotide residues. Class II mutated DNAs require higher Mg2+ concentrations than the wild-type DNA to be fully activated as factor Y ATPase effectors and cannot be stimulated in the ATPase reaction by monovalent salt at suboptimal levels of Mg2+. The implication of this mutant phenotype on the role of secondary and tertiary DNA structure in determining an active site is examined in the accompanying article (Soeller, W., Abarzúa, P., and Marians, K. J. (1984) J. Biol. Chem. 259, 14293-14300). Class III mutations coinactivate both the ATPase effector and DNA replication template activity of the site, indicating that they probably represent essential contact points between factor Y and the DNA. Class IV mutated DNAs behave in a manner similar to class II mutated DNAs in the ATPase reaction, but have a replication template activity intermediate between that of the class III and class II mutant DNAs. It is possible that these mutant DNAs are deficient in their ability to catalyze, during primosome assembly, a step subsequent to that of factor Y binding.(ABSTRACT TRUNCATED AT 400 WORDS)