ROLE OF SECONDARY STRUCTURE IN DETERMINING AN ACTIVE PRIMOSOME ASSEMBLY SITE

Abstract

Discrete, single-stranded DNA segments from 0X174 viral DNA strands and from the H (pas-BH) and L (pas-BL) strands of pBR322 and ColE1 DNAs specifically induce ATP hydrolysis by E. coli replication factor Y (protein n'). These same segments have the potential to form higher order DNA structures and function as assembly sites for a host-encoded, multi-enzyme complex (the primosome) whose components were previously shown to be required for priming DNA synthesis on 0X174 viral strands.;In order to identify important features of these primosome assembly sites with respect to their interaction with factor Y, I have studied the in vitro phenotypes of mutant pas sequences generated by in vitro mutagenesis procedures.;Characterization of the single base substitution mutations in pas-BL resolved them into two classes. Class I mutants exhibited wild-type levels of factor Y ATPase effector activity and origin function. These mutants represented either nonessential bases or regions acting as spacer sequences. Class II mutants were completely inactive as effectors for factor Y ATPase activity in the presence of low concentrations of Mg('2+). However, increasing Mg('2+) concentrations specifically restored the effector activity of these mutants. Mn('2+) and spermidine could substitute for Mg('2+) in producing this differential effect on class II mutant DNAs. Since these ligands are known to affect the stability of duplex DNA, these results implied that duplex DNA played some role in the interaction of factor Y with pas sequences.;With the intent of obtaining evidence of base pairing within pas-BL, multiple base substitution mutants and their revertants were examined. Mutant DNAs which had two or three base alterations in the pas-BL sequence were generally inactive both as factor Y ATPase effectors and as primosome-dependent, complementary strand origins of DNA replication (class III mutations). Revertants of these mutants were isolated using a plaque morphology assay. Some of these reversion events occurred in second sites within the pas sequence in a manner that strongly suggested that base pairing was essential among certain pas residues in the formation of an active primosome assembly site. These results indicated that the DNA sequence was important primarily for determining the compacted, higher order structure of a pas and that double helical, base-paired DNA played an important role in forming this higher order structure.