Complexes of Escherichia coli primase with the replication origin of G4 phage DNA.

Abstract

Escherichia coli primase (dnaG protein), an essential DNA replication enzyme, synthesizes a primer at the unique origin sequence of the single-stranded circular phage G4 DNA (Rowen, L., and Kornberg, A. (1978) J. Biol. Chem. 253, 758-764). Kinetic analyses suggest that for each DNA molecule at least two primase molecules participate in the reaction. Binding of 3H-labeled primase is specific for the G4 complementary strand origin region and is saturated at approximately 2 primase molecules/DNA circle. Such complexes, isolated by gel filtration, function in the absence of additional primase to convert the phage DNA to the duplex form. Although the primase-DNA complex is stable to refiltration, the DNA-bound enzyme can dissociate and reattach to function at the origin sequence of another G4 DNA circle. An antibody to primase blocks the action of primase in the free form or within a DNA complex and even interferes with extension of the primer by DNA polymerase III holoenzyme. These kinetic and binding studies of G4 priming, the least complicated of the primase systems, suggest that 2 primase molecules form a complex at the origin region and remain bound even after transcribing a sequence to prime DNA replication.