Purification and characterization of a mutant DnaB protein specifically defective in ATP hydrolysis.
The dnaB gene of Escherichia coli encodes an essential DNA replication enzyme. Fueled by the energy derived from the hydrolysis of ATP to ADP+P(i), this enzyme unwinds double-stranded DNA in advance of the DNA polymerase. While doing so, it intermittently stimulates primase to synthesize an RNA primer for an Okazaki fragment. To better understand the structural basis of these and other aspects of DnaB function, we have initiated a study of mutant DnaB proteins. Here, we report the purification and characterization of a mutant DnaB protein (RC231) containing cysteine in place of arginine at residue 231. The mutant protein attains a stable, properly folded structure that allows association of six promoters to form a hexamer, as is also true for wild-type DnaB. Further, the mutant protein interacts with ATP, the nonhydrolyzable ATP analog adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, and poly(dT), and it stimulates primase action. It is, however, profoundly deficient in ATP hydrolysis, helicase activity, and replication activity at the chromosomal origin of replication. In addition, while general priming reactions with wild-type DnaB and ATP elicited the synthesis of short primers, reactions with DnaB and ATP gamma S or with RC231 and either ATP or ATP gamma S stimulated the synthesis of significantly longer primers. On the basis of these observations, we suggest that primase interacts directly with DnaB throughout primer synthesis during general priming, until dissociation of DnaB from DNA or ATP hydrolysis by DnaB disrupts the interaction and leads to primer termination.
Shrimankar
P
P
Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4960.
Stordal
L
L
Maurer
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001120:Arginine; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D003545:Cysteine; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D019915:DNA Primase; D004261:DNA Replication; D053488:DnaB Helicases; D004926:Escherichia coli / Q000737:chemistry*; D005799:Genes, Dominant; D005804:Genes, Lethal; D006868:Hydrolysis; D009154:Mutation; D012316:RNA Nucleotidyltransferases / Q000187:drug effects
D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D001120:Arginine; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases; D053488:DnaB Helicases; D003545:Cysteine