title: Superhelical path of the DNA in the nucleoprotein complex that activates the initiation of phage phi 29 DNA replication.
abstract: Initiation of bacteriophage phi 29 DNA replication is activated by protein p6, a viral double-stranded DNA-binding protein that forms a nucleoprotein complex at the viral replication origins. This complex consists of a DNA right-handed superhelix wrapped around a multimeric protein p6 core with protein p6 dimers regularly bound every 24 base-pairs (bp). In this paper, we have constructed a concatemer formed by direct repeats of a 24 bp sequence previously proposed to act as a signal for protein p6 binding at a phi 29 replication origin. DNase I footprinting shows that protein p6 binds to the concatemer in a similar way to the phi 29 DNA replication origins but with higher affinity, indicating that the 24 bp sequence is a recognition signal for protein p6. Furthermore, the concatemer was cloned in a plasmid and, by electron microscopy, it was shown to be the highest-affinity protein p6 binding region present in the plasmid. Based on these observations, the linking number change restrained by protein p6 has been measured in a series of plasmids containing concatemers with different numbers of 24 bp repeats; from the values obtained the linking number change restrained by a single protein p6 dimer has been estimated (delta Lkd = 0.1). In addition, when protein p6-DNA complexes fixed with glutaraldehyde were analysed by electron microscopy, it was observed that protein p6 compacts 4.2-fold the length of naked DNA. These data, together with the previously known value of the surface-related DNA helical repeat in the complex (12 bp), completely define the superhelical path of the DNA in the complex: one superhelical turn approximately involves 63 bp and 2.6 protein p6 dimers, and the DNA superhelix has a diameter of 6.6 nm and a slope of 14 degrees. The data obtained also indicate that the DNA in the protein p6-DNA complex is undertwisted (11.5 bp/turn) and strongly bent (66 degrees/12 bp). These DNA conformational changes might contribute to the activation of phi 29 DNA initiation of replication by protein p6.
authors.affiliation: Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Serrano
authors.forename: C
authors.initials: C
authors.lastname: Gutiérrez
authors.forename: M
authors.initials: M
authors.lastname: Salas
authors.forename: J M
authors.initials: JM
authors.lastname: Hermoso
mesh_terms: D017103:Bacillus Phages / Q000235:genetics*; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004261:DNA Replication; D004278:DNA, Superhelical / Q000737:chemistry*; D004279:DNA, Viral / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003852:Deoxyribonucleoproteins / Q000737:chemistry*; D011564:Furocoumarins; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry*; D014779:Virus Replication
chemical_list: D003432:Cross-Linking Reagents; D004278:DNA, Superhelical; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D011564:Furocoumarins; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; C080198:viral protein p6