Published online 26 April 2004
Nucleic Acids Research, 2004, Vol. 32, No. 8 2306-2314
© 2004 Oxford University Press
Genome wide, supercoiling-dependent in vivo binding of a viral protein involved in DNA replication and transcriptional control
Instituto de Biología Molecular ‘Eladio Viñuela’ (CSIC), Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain
*To whom correspondence should be addressed. Tel: +34 91 4978435; Fax: +34 91 4978490; Email: msalas{at}cbm.uam.es
Received February 2, 2004; Revised March 26, 2004; Accepted April 4, 2004
Protein p6 of Bacillus subtilis bacteriophage 
29 is essential for phage development. In vitro it activates the initiation of DNA replication and is involved in the early to late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA forms a right-handed superhelix wrapping around a multimeric protein core. However, there was no evidence of p6 binding to 29 DNA in vivo. By crosslinking, chromatin immunoprecipitation and real-time PCR we show that protein p6 binds to most, if not all, the viral genome in vivo, although with higher affinity for both DNA ends, which contain the replication origins. In contrast, the affinity for plasmid DNA is negligible, but greatly increases when the negative supercoiling decreases, as shown in vivo by treatment of cells with novobiocin and in vitro by fluorescence quenching with plasmids with different topology. In conclusion, binding of protein p6 all along the 29 genome strongly suggests that its functions in replication and transcription control could be local outcomes of a more global role as a histone-like protein. The p6 binding dependence on DNA topology could explain its preferential binding to viral with respect to bacterial DNA, whose level of negative supercoiling is presumably higher than that of 29 DNA.
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