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Cleavage of the bacteriophage P1 packaging site (pac) is regulated by adenine methylation
1E. I. duPont de Nemours & Co. Inc., Central Research and Development Department, Wilmington, DE 19880-0328.
Abstract:
The packaging of bacteriophage PI DNA is initiated when the phage packaging site (pac) is recognized and cleaved and continues until the phage head is full. We have previously shown that pac is a 162-base-pair segment of P1 DNA that contains seven DNA adenine methyltransferase methylation sites (5'-GATC). We show here that cleavage of pac is methylation sensitive. Both in vivo and in vitro experiments indicate that methylated pac is cleavable, whereas unmethylated pac is not. Moreover, DNA isolated from P1 phage and containing an uncut pac site was a poor substrate for in vitro cleavage until it was methylated by the Escherichia coli DNA adenine methyltransferase. Comparison of that uncut pac DNA with other viral DNA fragments by digestion with methylation-sensitive restriction enzymes indicated that the uncut pac DNA was preferentially undermethylated. In contrast, virion DNA containing a cut pac site was not undermethylated. We believe these results indicate that pac cleavage is regulated by adenine methylation during the phage lytic cycle.
Insights
Bacteriophage P1 DNA packaging requires cleavage at the pac site. Methylation of this site by adenine methyltransferase is essential for cleavage, regulating DNA packaging during the phage lytic cycle.
Area of Science:
- Molecular Biology
- Virology
- Epigenetics
Background:
- Bacteriophage P1 DNA packaging is initiated by recognition and cleavage at the phage packaging site (pac).
- The pac site is a 162-base-pair DNA segment containing seven 5'-GATC methylation sites recognized by DNA adenine methyltransferase.
- Previous studies established the role of the pac site in DNA packaging.
Purpose of the Study:
- To investigate the role of DNA adenine methylation in the cleavage of the bacteriophage P1 pac site.
- To determine if pac site cleavage is sensitive to methylation status.
Main Methods:
- In vivo and in vitro experiments were conducted to assess pac site cleavage.
- Methylation-sensitive restriction enzyme digestion was used to compare viral DNA fragments.
- DNA adenine methyltransferase was used to methylate DNA in vitro.
Main Results:
- Cleavage of the pac site is sensitive to methylation; methylated pac is cleavable, while unmethylated pac is not.
- Uncut pac DNA from P1 phage was a poor substrate for in vitro cleavage until methylated by Escherichia coli DNA adenine methyltransferase.
- Unmenthylated pac DNA was preferentially undermethylated compared to other viral DNA fragments, while cut pac sites in virion DNA were not undermethylated.
Conclusions:
- Pac cleavage is regulated by adenine methylation during the bacteriophage P1 lytic cycle.
- DNA adenine methylation acts as a crucial control mechanism for initiating phage DNA packaging.