ANALYSIS OF FOUR DISTINCT CLASSES OF PRIMOSOME ASSEMBLY SITE MUTANTS (MUTAGENESIS, DNA REPLICATION)
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The primosome, a multi-enzyme complex, is responsible for priming of lagging-strand DNA synthesis in E. coli. One of the proteins of the primosome is E. coli replication factor Y. Recognition and binding of this site-specific SS DNA dependent ATPase to its effector sites is considered the initial event in primosome formation. In order to understand the interaction of factor Y with a primosome assembly site (pas), a new site-specific mutagenesis procedure and a novel fl phage recombinant DNA cloning vector were developed to generate a library of single base substitutions along the pBR322 H strand site (pas-BH). Forced misincorporation of an (alpha)-thiodeoxynucleoside triphosphate analog onto the 3'-OH of E. coli exonuclease III-treated heteroduplex templates targeted the mutagen to the region of interest. Asymmetric segregation of mutant sequences was accomplished by RF (--->) SS(c) DNA synthesis in vitro, initiated from the 0X174 viral strand origin sequence present on the vector DNA. Characterization of the mutated sites as effectors for factor Y ATPase activity and as templates for primosome-directed DNA synthesis yielded four distinct classes of mutants and revealed the crucial role played by mono-, di- and multivalent cations in the interaction of factor Y with the wild-type and mutant sites. Class I mutants displayed wild-type levels of both activities and they probably represent non-essential residues. Class II mutants exhibited up to two-fold reduction in their template activity for DNA synthesis and an altered requirement for Mg2+, spermidine and monovalent ions in their activity as factor Y ATPase effectors. Class III showed a several fold reduction in activity as a pas and as effectors for factor Y ATPase, even at higher concentrations of mono-, di- and polyvalent cations. Class IV mutants behaved similarly to class II mutants in the ATPase assay; however, when assayed as templates for DNA synthesis they were consistently three to five-fold less efficient than the wild-type site. The phenotype of the class II and class IV mutant DNAs strongly suggests that a pas is a complex higher order structure requiring specific secondary and tertiary interactions for correct folding into a functional structure. In addition, the replication template efficiency of class IV mutant DNAs suggests that these mutations may define contact points for some of the other primosomal proteins.
Source: Dissertation Abstracts International, Volume: 45-08, Section: B, page: 2399.