Cleavage of single-stranded DNA by the varphiX174 A protein: The A-single-stranded DNA covalent linkage.
Phage varphiX174 A(*) protein cleaves single-stranded DNA and then binds to the 5'-phosphorylated terminus of the cleaved DNA fragment, forming a covalent protein-DNA complex. The bound A(*) protein can religate the termini to form covalently closed single-stranded circles. To determine the nature of the covalent linkage and the amino acid involved, we used A(*) protein to cleave DNA synthesized in vivo with [alpha-(32)P]dATP to form the A(*)-single-stranded DNA complex. The complex was then digested with DNase I and the (32)P-labeled A(*) protein was isolated by electrophoresis on polyacrylamide gels. The isolated complex was digested with either trypsin or Pronase. Incubation of the tryptic digest with snake venom phosphodiesterase gave (32)P-labeled products that migrated on electrophoresis on cellulose plates to the cathode, indicating covalent linkage of (32)P-labeled dAMP residues to a tryptic peptide. High concentrations of snake venom phosphodiesterase released all of the (32)P label as free dAMP. Formic acid/diphenylamine depurination (Burton reaction) of the [alpha-(32)P]dATP-labeled peptide-oligonucleotide complexes caused a transfer of the labeled phosphate from dAMP to the peptide. The phosphorylated peptides were isolated on cellulose plates and shown to be sensitive to bacterial alkaline phosphatase, indicating that a phosphodiester bond linked the peptides to the dAMP. The phosphorylated product of the Pronase digest was identified as free phosphotyrosine by its mobility in three different chromatography systems. Likewise, acid hydrolysis (5.6 M HCl, 110 degrees C, 2 hr) of the phosphorylated tryptic peptides revealed linkage of the phosphate to a tyrosine. Thus, A(*) protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated terminus via a tyrosyl-dAMP phosphodiester bond.
Department of Biochemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
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Sanhueza
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Eisenberg