The role of template superhelicity in the initiation of bacteriophage λ DNA replication

Abstract

The prepriming steps in the initiation of bacteriophage .lambda. DNA replication depend on the action of the .lambda. O and P proteins and on the DnaB helicase, single-stranded DNA binding protein (SSB), and DnaJ and DnaK heat shock proteins of the E. coli host. The binding of multiple copies of the .lambda. O protein to the phage replication origin (ori.lambda.) initiates the ordered assembly of a series of nucleoprotein structures that form at ori.lambda. prior to DNA unwinding, priming and DNA synthesis steps. Since the initiation of .lambda. DNA replication is known to occur only on supercoiled templates in vivo and in vitro, we examined how the early steps in .lambda. DNA replication are influenced by superhelical tension. All initiation complexes formed prior to helicase-mediated DNA-unwinding form with high efficiency on relaxed ori.lambda. DNA. Nonetheless, the DNA templates in these structures must be negatively supertwisted before they can be replicated. Once DNA helicase unwinding is initiated at ori.lambda., however, later steps in .lambda. DNA replication processed efficiently in the absence of superhelical tension. We concluded that supercoiling is required during the initiation of .lambda. DNA replication to facilitate entry of a DNA helicase, presumably the DnaB protein, between the DNA strands.