Activation of Replication Origins in ϕ29-related Phages Requires the Recognition of Initiation Proteins to Specific Nucleoprotein Complexes

Raimundo Freire,Manuel Serrano,Margarita Salas,José M Hermoso

doi:10.1074/jbc.271.48.31000

Abstract

Protein p6 of Bacillus subtilis phage phi29 activates the initiation of viral DNA replication by forming a multimeric nucleoprotein complex at the origins of replication, located at both ends of the linear genome. This activation requires a precise positioning of the protein p6 array with respect to the initiation site. To investigate this activation mechanism, we have purified the phi29 protein p6 counterparts from the related phages Nf and GA-1 and analyzed the formation of complexes with DNA. In the homologous protein p6-DNA complexes the phi29 and Nf protein arrays showed an identical positioning, different than that of the GA-1 protein array. In contrast, in the heterologous complexes the protein showed a different arrangement except in the case of the Nf protein-phi29 DNA complex. We have also purified the proteins involved in the initiation of replication (terminal protein and DNA polymerase) from phages Nf and GA-1 and measured the ability of the different p6 proteins to activate homologous and heterologous replication origins. The results obtained indicate that the activation requires not only the formation of a specific nucleoprotein complex but also its specific recognition by the proteins involved in the initiation of DNA replication.

Highlights

Bacillus subtilis phage ␾29 has a linear, double-stranded DNA with a terminal protein (TP)1 covalently linked to the 5Ј ends
These results are consistent with a specific recognition of the protein p6 nucleoprotein complex by the proteins involved in the initiation of ␾29 DNA replication, namely TP and/or DNA polymerase
Preliminary studies showed that DNase I footprints of ␾29 protein p6 with Nf and GA-1 DNA terminal fragments were different than those observed with ␾29 DNA [17]

Summary

EXPERIMENTAL PROCEDURES

Nucleotides, Oligonucleotides, and Enzymes—Glutaraldehyde was obtained from Serva. The corresponding 32P-labeled DNA fragments (2 ng) were mixed with 500 ng of the corresponding viral DNA and incubated with the indicated amounts of the different proteins in 25 ␮l of a buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 20 mM ammonium sulfate, and 10% glycerol. The reaction mixture in the initiation of replication assay contained, in 25 ␮l, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 20 mM ammonium sulfate, 0.1 mg/ml bovine serum albumin, 1 mM dithiothreitol, 0.25 ␮M [␣-32P]dATP (0.25 ␮Ci), 125 ng each of ␾29 DNA polymerase and TP, ϳ6 fmol of the indicated DNA fragment or 500 ng of the corresponding TP-DNA, and the indicated amounts of the corresponding protein p6. The Fuji imaging plate type Bass IIIs and the Bioimaging analyzer BAS 1500 were used for quantitation

RESULTS

Nucleoprotein Complex That Activates the Replication Origin

DISCUSSION