Deletion mutants defining the Escherichia coli replication factor Y effector site sequences in pBR322 DNA.

The Escherichia coli DNA replication factor Y, along with other genetically undefined replication proteins, is involved in a dnaB-, dnaC-, and dnaG-dependent pathway of primer formation on phi X174 single-stranded circular DNA. In addition, replication factor Y has a site-specific, single-stranded DNA-dependent ATPase activity. We have previously demonstrated the presence of two factor Y effector sites on pBR322 DNA. When inserted into the filamentous phage f1R229, these sites can function as rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent origins of DNA replication. We report here the construction of deletion mutants of the two pBR322 factor Y effector sites. These deleted sites no longer function as effectors for factor Y ATPase activity nor as templates for rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent DNA synthesis. We conclude that the DNA sequences required for factor Y ATPase activity and origin function are likely to be identical.