Purification and characterization of DnaC810, a primosomal protein capable of bypassing PriA function.
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 phiX174 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.
Xu
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Biochemistry and Structural Biology Graduate Program, Cornell University Graduate School of Medical Sciences, New York, New York 10021, USA.
Marians
K J
KJ
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010584:Bacteriophage phi X 174 / Q000235:genetics*; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000821:virology; D029968:Escherichia coli Proteins; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D051716:Replication Protein A; D013489:Suppression, Genetic
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D051716:Replication Protein A; D004265:DNA Helicases; D053488:DnaB Helicases