J Biol Chem, Vol. 275, Issue 11, 8196-8205, March 17, 2000

Purification and Characterization of DnaC810, a Primosomal Protein Capable of Bypassing PriA Function^*

Liewei Xu and Kenneth J. Marians^§¶

From the  Biochemistry and Structural Biology Graduate Program, Cornell University Graduate School of Medical Sciences, New York, New York 10021 and the ^§ Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Escherichia coli strains lacking PriA are severely compromised in their ability to repair UV-damaged DNA and to perform homologous recombination. These phenotypes arise because of a lack of PriA-directed replication fork assembly at recombination intermediates such as D-loops. Naturally arising suppressor mutations in dnaC restore strains carrying the priA2::kan null allele to wild-type function. We have cloned one such gene, dnaC810, and overexpressed, purified, and characterized the DnaC810 protein. DnaC810 can support a PriA-independent synthesis of X174 complementary strand DNA. This can be attributed to its ability, unlike wild-type DnaC, to catalyze a SSB-insensitive general priming reaction with DnaB and DnaG on any SSB-coated single-stranded DNA. Gel mobility shift analysis revealed that DnaC810 could load DnaB directly to SSB-coated single-stranded DNA as well as to D loop DNA. This explains the ability of DnaC810 to bypass the requirement for PriA, PriB, PriC, and DnaT during replication fork assembly at recombination intermediates.