Identification of a region of Escherichia coli DnaB required for functional interaction with DnaG at the replication fork.
The fundamental activities of the replicative primosomes of Escherichia coli are provided by DnaB, the replication fork DNA helicase, and DnaG, the Okazaki fragment primase. As we have demonstrated previously, DnaG is recruited to the replication fork via a transient protein-protein interaction with DnaB. Here, using site-directed amino acid mutagenesis, we have defined the region on DnaB required for this protein-protein interaction. Mutations in this region of DnaB affect the DnaB-DnaG interaction during both general priming-directed and phiX174 complementary strand DNA synthesis, as well as at replication forks reconstituted in rolling circle DNA replication reactions. The behavior of the purified mutant DnaB proteins in the various replication systems suggests that access to the DnaG binding pocket on DnaB may be restricted at the replication fork.
Chang
P
P
Molecular Biology Graduate Program, Weill Graduate School of Medical Sciences of Cornell University and the Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Marians
K J
KJ
D019943:Amino Acid Substitution; D001426:Bacterial Proteins; D010584:Bacteriophage phi X 174 / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D053488:DnaB Helicases; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008958:Models, Molecular; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D004279:DNA, Viral; C022262:Okazaki fragments; D004247:DNA; D019915:DNA Primase; D004265:DNA Helicases; D053488:DnaB Helicases