In vivo DNA binding of bacteriophage GA-1 protein p6.
Bacteriophage GA-1 infects Bacillus sp. strain G1R and has a linear double-stranded DNA genome with a terminal protein covalently linked to its 5' ends. GA-1 protein p6 is very abundant in infected cells and binds DNA with no sequence specificity. We show here that it binds in vivo to the whole viral genome, as detected by cross-linking, chromatin immunoprecipitation, and real-time PCR analyses, and has the characteristics of a histone-like protein. Binding to DNA of GA-1 protein p6 shows little supercoiling dependency, in contrast to the ortholog protein of the evolutionary related Bacillus subtilis phage phi29. This feature is a property of the protein rather than the DNA or the cellular background, since phi29 protein p6 shows supercoiling-dependent binding to GA-1 DNA in Bacillus sp. strain G1R. GA-1 DNA replication is impaired in the presence of the gyrase inhibitors novobiocin and nalidixic acid, which indicates that, although noncovalently closed, the viral genome is topologically constrained in vivo. GA-1 protein p6 is also able to bind phi29 DNA in B. subtilis cells; however, as expected, the binding is less supercoiling dependent than the one observed with the phi29 protein p6. In addition, the nucleoprotein complex formed is not functional, since it is not able to transcomplement the DNA replication deficiency of a phi29 sus6 mutant. Furthermore, we took advantage of phi29 protein p6 binding to GA-1 DNA to find that the viral DNA ejection mechanism seems to take place, as in the case of phi29, with a right to left polarity in a two-step, push-pull process.
Alcorlo
Martín
M
Instituto de Biología Molecular Eladio Viñuela (CSIC), Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Salas
Margarita
M
Hermoso
José M
JM
D001407:Bacillus / Q000235:genetics / Q000821:virology; D017103:Bacillus Phages / Q000235:genetics* / Q000378:metabolism*; D004261:DNA Replication; D004278:DNA, Superhelical; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D009268:Nalidixic Acid; D009675:Novobiocin; D011485:Protein Binding; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004278:DNA, Superhelical; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C080198:viral protein p6; D009675:Novobiocin; D009268:Nalidixic Acid