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

The preptriming steps in the initiation of bactericphage λ DNA replication depend on the action of the λ 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 λ O protein to the phage replication origin (oriλ) initiates the ordered assembly of a series of nucleoprotein structures that form at oriλ prior to DNA unwinding, priming and DNA synthesis steps. Since the initiation of A DNA replication is known to occur only on supercoiled templates in vivoand in vitro, We examined how the early steps in A DNA replication are influenced by superhelical tension. All initiation complexes formed prior to helicase-mediated DNA-unwinding form with high efficiency on relaxed oriλ DNA. Nonetheless, the DNA templates in these structures must be negatively super-twisted before they can be replicated. Once DNA helicase unwinding is initiated at oriλ, however, later steps in A DNA replication proceed efficiently in the absence of superhelical tension. We conclude that supercoiling is required during the initiation of A DNA replication to facilitate entry of a DNA helicase, presumably the DnaB protein, between the DNA strands.