Assembly of phage phi 29 genome with viral protein p6 into a compact complex.
The formation of a multimeric nucleoprotein complex by the phage phi 29 dsDNA binding protein p6 at the phi 29 DNA replication origins, leads to activation of viral DNA replication. In the present study, we have analysed protein p6-DNA complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. Under conditions that greatly favour protein-DNA interaction, the saturated phi 29 DNA-protein p6 complex appears as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex.
Gutiérrez
C
C
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
Freire
R
R
Salas
M
M
Hermoso
J M
JM
D017103:Bacillus Phages / Q000235:genetics*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D005363:Ficusin / Q000494:pharmacology; D016679:Genome, Viral; D005976:Glutaral; D008836:Micrococcal Nuclease; D011485:Protein Binding; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C080198:viral protein p6; D008836:Micrococcal Nuclease; D005363:Ficusin; D005976:Glutaral