Fragmentation of Bacillus bacteriophage phi105 DNA by complementary single-stranded DNA in the cohesive ends of the molecule

Abstract

The structure of DNA from the temperate Bacillus subtilis phage 4105 was examined by using the restriction endonuclease EcoRI and by sedimentation analysis. The DNA contains six EcoRI cleavage sites. Although eight DNA fragments were identified in the EcoRI digests, the largest of these was shown to consist of the two fragments that carry the cohesive ends of the phage DNA. In neutral gradients, the majority of whole 4105 DNA sedimented as nicked circles and the remainder as oligomers. No unit-length linear structures were detected. The associated cohesive ends could be sealed by DNA ligase from Escherichia coli and could be cleaved by S1 nuclease. On the basis of these results and previously reported studies, it appears that, as isolated from phage particles, 4105 DNA is a circular molecule that is formed from the linear structure by the association of complementary single-stranded DNA. 377

Correlated genetic and EcoRI cleavage map of Bacillus subtilis bacteriophage phi105 DNA

“…First, while the A att site, through which integration into the host chromosome occurs, is situated in the midregion of a cyclized A genome (for review, see reference 8), the 4105 att site involves the ends of the phage DNA (1,5). 4105 DNA, like A, can be reversibly cyclized (2,4), but unlike A, 4105 DNA is circular as extracted from the phage (16). Whether the ends of 4105 DNA act independently during integration or in concert, as would be the case for the circular form, is still an open question.…”

“…As part of the further genetic analysis of 4105, we wished to develop a correlated physical and genetic map of the phage chromosome. In an earlier study (16), eight EcoRI-generated DNA fragments, A through H, were identified. Two of these fragments, C and D, were shown to be derived from the ends of the molecule, D from the left and C from the right end, respectively.…”

“…The Ksus7 mutation blocks 4105 DNA replication (1), and this mutant, in contrast to the other 105sus mutants, cannot be rescued efficiently from an suhost. All other strains used for the induction and propagation of phage have been previously described (16).…”

“…Preparation of phage DNA. All the phage DNAs used were phenol extracted from CsCl-purified phage by using previously described methods and strains (16). The specific activity of the 32P-labeled 4105 DNA was 30,000 cpm/jig.…”

“…Agarose gel electrophoresis. Analytical agarose gels were run as previously described (16). Preparative agarose gel electrophoresis was performed in a similar manner with the following modifications.…”