Mechanism of dnaB protein action. III. Allosteric role of ATP in the alteration of DNA structure by dnaB protein in priming replication.

dnaB protein binds ATP in a binary complex containing 1 nucleotide/protomer with a dis- sociation constant (KD) near 1 x 10-M.Other ribonu- cleoside triphosphates, deoxyribonucleoside triphos- phates, and ADP compete for the same site with similar affinities.The nonhydrolyzed analog, adenosine-5'-0- (3'-thiotriphosphate), binds to the site far more tightly (KD near 1 X 10-7 M), but still exchanges very rapidly with free nucleotide.dnaB protein does not form a strong binary complex with single-stranded (SS) DNA, but forms a ternary complex with SS DNA and ATP.The latter complex is stable enough for isolation only when ATP is replaced by adenosine-5'-0-(3'-thiotriphosphate), thus demonstrating the allosteric role of the nucleotide in increasing the affinity of dnaB protein for SS DNA.The ternary complex (dnaB protein-SS DNA.ATP) can also be stabilized by binding of dnaC protein or primase.A distributive (nonprocessive) mechanism for dnaB protein in its promotion of priming is predicated on its release from DNA by hydrolysis of ATP.The influence of dnaB protein on DNA struc- ture can be inferred from the effects of DNA intercalating agents.Ethidium bromide, for example, profoundly inhibits priming by the dnaB protein-primase system on polythymidylate.Enhancement of ethidium bromide fluorescence by polythymidylate depends on dnaB pro- tein and ATP, implying a ternary complex with secondary structure.In its interaction with SS DNA, the dnaB protein hexamer covers -80 nucleotide residues as in- ferred from protection of DNA from nucleases.These studies all suggest that the allosteric role of ATP is expressed in a ternary complex with SS DNA and dnaB protein, strengthened by dnaC protein or primase, in which dnaB protein "engineers." a secondary structure from suitable sequences in SS DNA for recognition by primase.Formation of multiple primers on single-stranded DNA not coated by single-stranded DNA binding protein depends on the joint action of dnaB protein and primase (1).The system