SUPERHELICAL PATH OF THE DNA IN THE NUCLEOPROTEIN COMPLEX THAT ACTIVATES THE INITIATION OF PHAGE-PHI-29 DNA-REPLICATION

Initiation of bacteriophage φ29 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 regularlybound every 24 base-pairs (bp). In this paper, we have constructed a concatemer formed by directrepeats of a 24 bp sequence previously proposed to act as a signal for protein p6 binding at a φ29 replication origin. DNase I footprinting shows that protein p6 binds to the concatemerin a similar way to the φ29 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 changerestrained 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 restrainedby a single protein p6 dimer has been estimated (ΔLkd = 0·1). In addition, when protein p6-DNA complexes fixed with glutaraldehyde were analysed by electron microscopy, it was observed that protein p6 compacts4·-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 pathof 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°. The data obtained also indicate that the DNA in the protein p6-DNA complex is undertwisted (11·5 bp/turn) and strongly bent (66°/12 bp). These DNA conformational changes might contribute to the activation of φ29 DNA initiation of replication by protein p6. © 1993 Academic Press, Inc.