A* protein of bacteriophage φX174 carries an oligonucleotide which it can transfer to the 3−OH of a DNA chain

Abstract

The bacteriophage φX174 gene A encodes two proteins: gene A protein and A* protein. Purified A* protein acts as a single‐stranded, DNA‐specific endonuclease which remains covalently attached to the 5′‐end of the cleavage site. Incubation of A* protein with the synthetic heptamer CAACTTG or with oligonucleotides which yield this heptamer after cleavage with the A* protein yields oligonucleotides with the sequences CAACTTGAG, CAACTTGAGG and CAACTTGAGGA. This indicates that A* protein carries an oligonucleotide with the sequence ‐AG, AGG or ‐AGGA. The oligonucleotide can be transferred to the 3′‐end of the heptamer CAACTTG. This suggests that A* protein reacts with a specific DNA sequence in the infected cell.

The bond in the bacteriophage øX174 gene A protein‐DNA complex is a tyrosyl‐5'‐phosphate ester

“…It has retained a number of enzymatic activities of gene A protein [ 11,121. A portion of the A* protein molecules in preparations of purified A* protein carry a covalently linked specific oligonucleotide, suggesting that A* protein performs a cleavage reaction during phage development [13]. (60 pmol), 1.5 ~1 of 0.75 mM dithiothreitol (DTT) and 3.75 ~1 of 20 mM ATP were added and the mixture (total volume 85 ~1) was incubated for I b at 4°C to anneal the heptamer and the undecamer to the hexadecamer.…”

Mutational analysis of the bacteriophage φX174 replication origin

“…This part of the gene A protein is responsible for the specificity of the cleavage of double-stranded DNA and not for the cleavage reaction per se. A* protein, which lacks the N-terminal third of the polypeptide chain of gene A protein, also cleaves and ligates single-stranded DNA, but is unable to cleave double-stranded DNA (Langeveld et al, 1979(Langeveld et al, , 1981Eisenberg & Finer, 1980;Van Mansfeld et al, 1982). The two tyrosyl residues involved in the cleavage-ligation reaction of gene A protein have been determined in the Cterminal part of the gene A protein (Van Mansfeld et al, 1986).…”

DNA replication of single-stranded Escherichia coli DNA phages

“…Alternatively, the A* protein, which is a rather unspecific single-stranded nuclease, may cleave single-stranded regions in the replication fork and may affect in this way double-stranded DNA replication [118,119]. Evidence for this view may be derived from the observation that part of the A* molecules isolated "from infected cells carries an oligonucleotide [123]. The covalently bound oligonucleotide is a remnant of an earlier cleavage reaction within the cell.…”

“…The nucleotide sequence of this oligonucleotide, AGGATAA (Ref. 123; and A.D.M. Van Mansfeld, unpublished results), strongly suggests that it originated from cleavage by A* protein of the second gene A protein cleavage site TTACTTG ~AGGATAA on ~X DNA (~X 984-997) [91].…”

“…As described above, a role for the A* protein in the shutdown of stage II replication has been suggested by the DNA binding and nuclease properties of the protein [118][119][120][121][122][123]. A positive role for the A* protein in the switch from stage II to stage III replication such as described for the gene X protein in the filamentous phage cannot be ruled out [140].…”