Effect of SSB protein on cleavage of single-stranded DNA by phi X gene A protein and A* protein.

AUTOR(ES)

van Mansfeld, A D

RESUMO

Gene A protein of bacteriophage phi X174 plays a role as a site-specific endonuclease in the initiation and termination of phi X rolling circle DNA replication. To clarify the sequence requirements of this protein we have studied the cleavage of single-stranded restriction fragments from phi X and G4 viral DNAs using purified gene A protein. The results show that in both viral DNAs cleavage occurs at the origin and at one additional site which shows striking sequence homology with the origin region. During rolling circle replication the single-stranded viral DNA tail is covered with single-stranded DNA binding (SSB) protein. Therefore, we have also studied the effect of SSB on phi X gene A protein cleavage. In these conditions only single-stranded fragments containing the complete or almost complete origin region of 30 bases are cleaved, whereas cleavage at the additional sites of phi X or G4 viral DNAs does not occur. A model for termination of rolling circle replication which is based on these findings is presented. Finally, we present evidence that the second product of gene A, the A* protein, cleaves phi X viral DNA at the additional cleavage site in the presence of SSB, not only in vitro but also in vivo. The functional significance of this cleavage in vivo is discussed.

Documentos Relacionados

• Cleavage and circularization of single-stranded DNA: a novel enzymatic activity of phi X174 A* protein.
• Cleavage of single-stranded DNA by the A and A* proteins of bacteriophage phi X174.
• Cleavage of single-stranded DNA by the ϕX174 A* protein: The A*-single-stranded DNA covalent linkage
• Cleavage of phi X174 single-stranded DNA by gene A protein and formation of a tight protein-DNA complex.
• Cleavage of single-stranded DNA by plasmid pT181-encoded RepC protein.