Superhelical path of the DNA in the nucleoprotein complex that activates the initiation of phage phi 29 DNA replication.
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.
Serrano
M
M
Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain.
Gutiérrez
C
C
Salas
M
M
Hermoso
J M
JM
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
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