Abstract
Escherichia coli DnaA protein initiates DNA replication from the chromosomal origin, oriC, and regulates the frequency of this process. Structure-function studies indicate that the replication initiator comprises four domains. Based on the structural similarity of Aquifex aeolicus DnaA to other AAA+ proteins that are oligomeric, it was proposed that Domain III functions in oligomerization at oriC (Erzberger, J. P., Pirruccello, M. M., and Berger, J. M. (2002) EMBO J. 21, 4763-4773). Because the Box VII motif within Domain III is conserved among DnaA homologues and may function in oligomerization, we substituted conserved Box VII amino acids of E. coli DnaA with alanine by site-directed mutagenesis to examine the role of this motif. All mutant proteins are inactive in initiation from oriC in vivo and in vitro, but they support RK2 plasmid DNA replication in vivo. Thus, RK2 requires only a subset of DnaA functions for plasmid DNA replication. Biochemical studies on a mutant DnaA carrying an alanine substitution at arginine 281 (R281A) in Box VII show that it is inactive in in vitro replication of an oriC plasmid, but this defect is not from the failure to bind to ATP, DnaB in the DnaB-DnaC complex, or oriC. Because the mutant DnaA is also active in the strand opening of oriC, whereas DnaB fails to bind to this unwound region, the open structure is insufficient by itself to load DnaB helicase. Our results show that the mutant fails to form a stable oligomeric DnaA-oriC complex, which is required for the loading of DnaB.
Highlights
Chromosomal DNA replication is initiated by DNA-binding proteins, which first recognize replication origins and assemble the enzymatic machinery that functions at each replication fork
Because the Box VII motif within Domain III is conserved among DnaA homologues and may function in oligomerization, we substituted conserved Box VII amino acids of E. coli DnaA with alanine by site-directed mutagenesis to examine the role of this motif
Box VII Mutants Are Inactive in Initiation from oriC—Despite showing that R281A is active in unwinding oriC and in binding to ATP and the DnaA boxes of oriC, we found that the mutant protein was inert in DNA replication of an oriC-containing plasmid in vitro (Fig. 5A)
Summary
Because the unwound region of oriC is speculated to be sufficient for the binding of two hexamers of DnaB from the DnaB-DnaC complex [24], mutant proteins should be active in DNA replication. To test these predictions, we substituted each of the strictly conserved amino acids in Box VII with alanine by site-directed mutagenesis (see “Experimental Procedures” and Fig. 1). Further biochemical characterization of R281A revealed that its inactivity in initiation is not a result of defects in binding to DnaA box sequences in oriC or oriV of the plasmid RK2 or to DnaB in the DnaB-DnaC complex. We show that the Box VII function is apparently unnecessary for initiation at the replication origin of RK2, supporting the notion that a subset of DnaA functions is needed at this plasmid origin
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