Gene A Protein of Bacteriophage ΦX174 is a Highly Specific Single-Strand Nuclease and Binds Via a Tyrosyl Residue to DNA After Cleavage
The sequence specificity of the endonuclease activity of gene A protein and A* protein was studied using synthetic oligonucleotides containing (part of) the sequence of the origin of ΦX RF DNA replication and single-stranded (ss) DNA fragments of ΦX and G4. From a comparison of the sequences that are cleaved a consensus sequence for cleavage of ssDNA by gene A protein has been deduced. This consensus sequence occurs in ssDNA of both ΦX and G4 at the origin and at one additional site. This is surprising since the rolling circle mechanism demands that gene A protein cleaves at the origin only. However, it could be shown that in the presence of SSB protein the ssDNAs of ΦX and G4 are only cleaved at the origin, which is probably due to a strong gene A protein binding site, the key sequence, which forms part of the 30 b. p. origin region of ΦX and related bacteriophages.
Gene A protein and A* protein bind covalently to the DNA at the 5’-end of the cleavage site. Using a uniquely, internally 32P- labelled oligonucleotide as a substrate, it was shown that gene A protein and A* protein are bound via a tyrosyl residue to the 5’-phosphate of the phosphodiester bond which is cleaved.
KeywordsProtein Binding Site Synthetic Oligonucleotide Phosphodiester Bond Origin Region Rolling Circle
Unable to display preview. Download preview PDF.
- 6.Baas, P. D., Heidekamp, F., Van Mansfeld, A. D. M., Jansz, H. S., Van der Marel, G. A., Veeneman, G. H. and Van Boom, J. H. (1981) in: “ICN-UCLA Symposia on Molecular and Cellular Biology: The Initiation of DNA Replication”, Vol. XXI, D. S. Ray and C. F. Fox, eds., Academic Press, New York.Google Scholar
- 12.Brown, D. R., Hurwitz, J., Reinberg, D. and Zipursky, S. L. (1982) in: “Nucleases”, S. M. Linn and R. J. Roberts, eds., Cold Spring Harbor Labroatory, Cold Spring Harbor, p. 187.Google Scholar
- 15.Kowalczykowski, S. C., Bear, D. G. and Von Hippel, P. H. (1981) in: “The Enzymes”, Vol. XVI, S. Boyer, ed., Academic Press, New York, p. 373.Google Scholar