Hyperinitiation of DNA replication in Escherichia coli leads to replication fork collapse and inviability.
Elevated dnaA expression from a multicopy plasmid induces more frequent initiation from the Escherichia coli replication origin, oriC, but viability is maintained. In comparison, chromosomally encoded dnaAcos also stimulates initiation, but this is lethal. By quantitative methods, we show that the level of initiation induced by elevated dnaA expression leads to collapsed replication forks that are mostly within 10 map units of oriC. Because forks collapse randomly, nucleoprotein complexes at specific sites such as datA are not the cause. When replication restart is blocked by a mutation in recB or priA, the increased initiations via elevated dnaA expression causes inviability. The amount of collapsed forks is substantially higher under elevated expression of dnaAcos compared to that of dnaA. We propose that the lethal phenotype of chromosomally encoded dnaAcos is a result of hyperinitiation that overwhelms the repair capacity of the cell.
Simmons
Lyle A
LA
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.
Breier
Adam M
AM
Cozzarelli
Nicholas R
NR
Kaguni
Jon M
JM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004261:DNA Replication / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D004927:Escherichia coli Infections / Q000276:immunology; D007700:Kinetics; D051379:Mice; D016254:Mutagenesis, Insertional; D010641:Phenotype; D016133:Polymerase Chain Reaction; D018741:Replication Origin; D017384:Sequence Deletion; D013601:T-Lymphocytes / Q000276:immunology
D017931:DNA Primers