Cleavage of single-stranded DNA by the ϕX174 A ^* protein: The A ^* -single-stranded DNA covalent linkage

Abstract

Phage fX174 A* protein cleaves singlestranded 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 vitro with [a-32P]dATP to form the A*-single-stranded DNA complex. The complex was then digested with DNase I and the 32P-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 32P-labeled products that migrated on electrophoresis on cellulose plates to the cathode, indicating covalent linkage of 32P-labeled dAMP residues to a tryptic peptide. (18)(19)(20). The isolation of an active A*-ss DNA complex in which the bound protein can religate the 5' and 3' termini to produce covalently closed singlestranded circles (19) and the fact that the 5' ends of the complex were blocked to the action of bacterial alkaline phosphatase and polynucleotide kinase (20) suggest that the A* protein binds covalently to the 5'-phosphorylated ends of the cleaved DNA. In the experiments described here, we elucidated the covalent linkage between the A* protein and the 5'-phosphorylated terminus of the cleaved ss DNA fragment. MATERIALS AND METHODSEnzymes. Snake venom phosphodiesterase, DNase I, and bacterial alkaline phosphatase were from Worthington. Bacterial alkaline phosphatase was further purified as described (21). The A* protein was purified from 4X174-infected E. coli cells as a by-product of the purification of OX gene A protein (10). Steps 1-5 were as described (10). The fraction that did not bind to hydroxyapatite contained the A* protein. This fraction was further purified through a heparine-Sepharose column. The protein was assayed by its ability to cleave ss DNA (18,19), the capacity to inhibit the OX stage II reaction in vitro (17), and by its mobility as a 34-to 36-kDa protein on polyacrylamide gels. Details of the purification scheme will be published elsewhere.Synthesis of [a-32P] 4285The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Bacteriophage phi X174 A protein cleaves single-stranded DNA and binds to it covalently through a tyrosyl-dAMP phosphodiester bond

“…Thus, in the A-ssDNA complex, the A protein is linked to DNA by a tyrosyl-dAMP phosphodiester bond. Similar results were obtained with the 0X A* protein (18), which is homologous in amino acid sequence to the C-terminal part of the A protein (14). Hence, the active sites for the endonucleolytic activity of A appear to reside within the C-terminal half of the protein.…”

“…The A-ssDNA complex was first digested with DNase I, isolated on a polyacrylamide gel, and finally treated extensively with trypsin. Details of the enzyme reactions have been described previously (7,18). The tryptic peptides were incubated with formic acid and diphenylamine as described by Burton (3).…”

“…The complexes were first digested with DNase I and subsequently electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. The DNase I treatment and electrophoresis were carried out essentially as previously described (7,18). The 32P-labeled A protein was identified by autoradiography as a band (lane 1) that comigrated with free A protein, which was electrophoresed in a separate lane and visualized by Coomassie blue staining (lane 2).…”

DNA Topoisomerases: Enzymes That Control DNA Conformation

“…This A * protein has been shown to form a covalent complex through a tyrosyl-dAMP phosphodiester bond (SANHUEZA and EISENBERG 1984).…”

Nonhomologous recombination in the parvovirus chromosome: role for a CTATTTCT motif.

“…6,1986 on known, but it appears to be unrelated to any of the known viral proteins (3,10). Since it remains bound to the 5' side of a DNA break and is attached to its DNA substrate via a phosphotyrosyl residue, its role in MVM DNA replication has been likened to that of the gene-A protein of 4X174 (3,14,24). Our results suggest that the Mr 60,000 topoisomerase I-like protein is also important in nonhomologous recombination, since its preferred cleavage site is associated with the recombination borders in deletion variants of the MVM chromosome and with the ends of broken viral chromosomes.…”