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P1 plasmid replication requires methylated DNA
1Laboratory of Chromosome Biology, BRI-Basic Research Program, NCI-Frederick Cancer Research Facility, MD 21701.
Abstract:
Plasmids driven by the plasmid replication origin of bacteriophage P1 cannot be established in Escherichia coli strains that are defective for the DNA adenine methylase (dam). Using a composite plasmid that has two origins, we show that the P1 origin cannot function even in a plasmid that is already established in a dam strain. An in vitro replication system for the P1 origin was developed that uses as a substrate M13 replicative-form DNA containing the minimal P1 origin. The reaction mixture contains a crude extract of E. coli and purified P1 RepA protein. In addition to being RepA dependent, synthesis was shown to be dependent on methylation of the dam methylase-sensitive sites of the substrate DNA. As the P1 origin contains five such sites in a small region known to be critical for origin function, it can be concluded that methylation of these sites is a requirement for initiation. This suggests that the postreplicational methylation of the origin may control reinitiation and contribute to the accuracy of the highly stringent copy-number control of the origin in vivo.
Insights
Bacteriophage P1 plasmid replication requires DNA adenine methylase (dam) activity. Methylation of dam-sensitive sites within the P1 origin is essential for initiation, controlling copy number.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Plasmids are crucial tools in molecular biology and genetics.
- Bacteriophage P1 plasmids are widely used for their stringent copy-number control.
- DNA adenine methylase (dam) is a key enzyme in bacterial DNA methylation.
Purpose of the Study:
- To investigate the role of DNA adenine methylase (dam) in the replication of bacteriophage P1 plasmids.
- To elucidate the mechanism by which the P1 origin initiates replication.
- To understand the basis of the stringent copy-number control of P1 plasmids.
Main Methods:
- Utilizing a composite plasmid with two origins of replication in Escherichia coli strains.
- Developing an in vitro replication system for the P1 origin using M13 replicative-form DNA.
- Employing crude E. coli extracts and purified P1 RepA protein in the in vitro system.
Main Results:
- Plasmids with the P1 origin cannot be established in dam-defective E. coli.
- The P1 origin fails to function even in established plasmids in dam strains.
- In vitro replication is dependent on RepA protein and methylation of dam-sensitive sites.
- Five dam-sensitive sites within the P1 origin are critical for initiation.
Conclusions:
- DNA adenine methylase (dam) activity and methylation of specific sites are essential for P1 origin function.
- Methylation is required for the initiation of P1 plasmid replication.
- Post-replicational methylation likely controls reinitiation and contributes to P1's stringent copy-number control.