Structure of the linkage between adenovirus DNA and the 55,000 molecular weight terminal protein

The structure of the protein-DNA linkage is determined and it is suggested that Ad5 DNA is bound to the terminal protein by a phosphodiester linkage to the β-OH of a serine residue and the 5′- OH of the terminal deoxycytidine residue of the DNA.

Abstract

The 5′ terminus of each complementary strand of adenovirus DNA isolated from virions is covalently linked to a protein with an apparent molecular weight of 55,000. We have determined the structure of the protein-DNA linkage. The 55,000 Mr protein, linked to a small [32P]oligonucleotide, was isolated after DNase digestion of uniformly 32P-labeled adenovirus 5 (Ad5) DNA-protein complex. The protein was digested with trypsin and the resulting [32P] peptides were analyzed with the following results. (1) Acid hydrolysis released a single phosphorylated amino acid which was identified as O-phosphoserine in four separate electrophoretic or chromatographic systems; (2) treatment with snake venom phosphodiesterase yielded exclusively dAMP, dCMP and dTMP as expected (there are no guanylate residues in the first 25 nucleotides at the 5′ ends of Ad5 DNA); (3) prior treatment of the [32P]peptide preparation with snake venom phosphodiesterase greatly reduced the yield of O-phosphoserine upon subsequent acid hydrolysis. These results suggest that Ad5 DNA is bound to the terminal protein by a phosphodiester linkage to the β-OH of a serine residue. This conclusion is supported by the finding that the DNA-protein linkage is readily hydrolyzed in alkali. In 50 mm-NaOH at 70 °C the half time for hydrolysis of the linkage is about ten minutes. After incubation of Ad5 DNA under these conditions we were able to label the 5′ termini with 32P by sequential treatment with alkaline phosphatase and polynucleotide kinase. Digestion of the end-labeled DNA to 5′ mononucleotides yielded [32P]dCMP. We conclude that the terminal protein is bound to Ad5 DNA by a phosphodiester linkage between the β-OH of a serine residue of the protein and the 5′-OH of the terminal deoxycytidine residue of the DNA.