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Escherichia coli dnaA initiation function is required for replication of plasmids derived from coliphage lambda.
Journal of Molecular Biology
|November 20, 1987
Summary
The dnaA gene is not essential for phage lambda growth or in vitro plasmid replication. However, in vivo, dnaA protein is crucial for normal lambda plasmid replication, likely by cooperating with RNA polymerase.
Area of Science:
- Molecular Biology
- Microbiology
- Virology
Background:
- The dnaA gene is essential for initiating DNA replication in Escherichia coli from oriC.
- Phage lambda growth and in vitro replication of its derived plasmids do not typically require dnaA gene function.
Purpose of the Study:
- To investigate the in vivo role of the dnaA gene in the replication of phage lambda plasmids.
- To elucidate the mechanism by which dnaA influences lambda plasmid replication in Escherichia coli.
Main Methods:
- Utilizing dnaA temperature-sensitive mutants (dnaAts) in Escherichia coli to study lambda plasmid replication.
- Assessing replication under different conditions, including the addition of rifampicin, to infer protein involvement.
Main Results:
- In vivo, lambda plasmid replication is dnaA-dependent, even though dnaA is dispensable for phage growth and in vitro replication.
- Inactivation of dnaA allows a single round of replication, with both normal and abortive replication being rifampicin-sensitive, indicating RNA polymerase involvement.
- Rifampicin resistance in specific dnaAts mutants suggests an interaction between DnaA protein and RNA polymerase during replication initiation.
Conclusions:
- DnaA protein likely co-operates with RNA polymerase to initiate replication at the ori lambda site in vivo.
- The dispensability of DnaA in certain contexts highlights the complex regulation of viral and plasmid DNA replication.