Genome-linked protein associated with the 5' termini of bacteriophage phi29 DNA

Abstract

A DNA-protein complex was isolated from Bacillus subtilis bacteriophage 429 by sucrose gradient sedimentation or gel filtration in the presence of agents known to break noncovalent bonds. A 28,000-dalton protein was released from this complex by subsequent hydrolysis of the DNA. The DNA-protein complex was examined for its susceptibility to enzymes which act upon the 5' and 3' termini of DNA molecules. It was susceptible to exonucleolytic degradation from the 3' termini by exonuclease III but not from the 5' termini by X exonuclease. Attempts to label radioactively the 5' termini by phosphorylation with T4 polynucleotide kinase were unsuccessful despite prior treatment with alkaline phosphatase or phosphatase treatment of denatured DNA. Removal of the majority of the bound protein by proteolytic digestion did not increase susceptibility. These results suggest that the linked protein is covalently attached to the 5' termini of 4)29 DNA.

VirD proteins of Agrobacterium tumefaciens are required for the formation of a covalent DNA-protein complex at the 5′ terminus of T-strand molecules.

“…The formation of similar DNA-protein complexes has been demonstrated for virus, phage and plasmid relaxation complex (Blair and Helinski, 1975;Lovett and Helinski, 1975;Rekosh et al, 1977;Yehle, 1978). In all cases the Fig.…”

“…As can be seen on Figure 2, only pronase treatment allowed us to detect the T-strand signal in the Southern blot analysis. The fact that treatment with different chaotropic agents as well as electrophoreses in the presence of 0.1 % SDS do not release the molecule is indicative of a covalent DNA -protein interaction (Guiney and Helinski, 1975;Rekosh et al, 1977;Yehle, 1978). It was also observed that the T-strand -protein complex remained stable when alkaline sucrose gradient was applied to purify the molecules (A.Herrera-Estrella et al, in preparation) as has been demonstrated previously for other DNA-protein complexes (Eisenberg and Denhardt, 1974).…”

“…Treatment with chaotropic agent and proteinase K digestion in situ Treatment of DNA -protein complexes with various chaotropic agents was as described (Lee et al, 1977;Harding et al, 1978;Ito, 1978;Yehle, 1978). In general, -500 ng of total DNA was incubated with different chaotropic agents in 200 Al volume at 37°C for 40 min.…”

Hairpin plasmid--a novel linear DNA of perfect hairpin structure.

“…The terminal proteins and inverted repetitions may be important in the replication of the linear DNA. Apart from the linear plasmids, similar terminal structures of linear DNA have been known in adenovirus (Rekosh et al, 1977;Carusi, 1977;Desiderio and Kelly, 1981;Steenbergh et al, 1977;Arrand and Roberts, 1979;Shinagawa and Padmanabhan, 1979) and Bacillus subtilis bacteriophage )29 (Salas et al, 1978;Harding et al, 1978;Yehle, 1978;Ito, 1978;Yoshikawa et al, 1981;Escarmis and Salas, 1981). The replication mechanisms of these viral linear DNAs have been extensively studied (Challberg and Kelly, 1982;Lichy et al, 1983;Stillman, 1983;Inciarte et al, 1980;Harding and Ito, 1980;Sogo et al, 1982;Prieto et al, 1984).…”

Cowpea mosaic virus VPg: sequencing of radiochemically modified protein allows mapping of the gene on B RNA

“…A large number of viruses contain a virus-coded protein covalently linked to the 5' terminus of the viral nucleic acid. These include DNA bacteriophages (Salas et al, 1978;Yehle, 1978;Harding et al, 1978;Geidushek and Ito, 1982;Bamford et al, 1983), animal DNA viruses (Rekosh et al, 1977;Keegstra et al, 1977;Gerlich and Robinson, 1980;Ganem et al, 1982;Molnar-Kimber et al, 1983;, animal RNA viruses (Lee et al, 1977;Flanegan et al, 1977;Burroughs and Brown, 1978;Hruby and Roberts, 1978;Golini et al, 1978;Sangar et al, 1977;Revet and Delain, 1982;Wimmer, 1982;Strauss and Strauss, 1983) and plant RNA viruses (Stanley et al, 1978;Daubert et al, 1978;Daubert and Bruening, 1979;Ghosh et al, 1979;Mayo et al, 1979Mayo et al, , 1982Hari, 1981; Reisman and De Zoeten, 1982;Koenig and Fritsch, 1982;Strauss and Strauss, 1983).…”