Mutational and functional analysis of the E. coli molecular chaperone, DnaJ, in the recognition of a lambda nucleoprotein complex

Abstract

Initiation of λ phage replication involves a series of sequential steps leading to the formation of a pre-replication complex (preRC) on λ DNA. However, replication can not proceed until this multimeric complex, composed of λO, λP and the E. coli DNA replicative helicase, DnaB, is recognized and subsequently remodeled by the molecular chaperones DnaJ and DnaK. Little is known about the mechanism by which the chaperones mediate the reorganization of this complex. Evidence indicates that DnaJ first recognizes the preRC and recruits DnaK through the stimulation of DnaK's intrinsic ATPase activity. This leads to partial disassembly of the preRC and subsequent loading of DnaB onto DNA. Unfortunately, the stoichiometry of the DnaJ:preRC binding reaction as well as the site(s) of their interaction remain enigmatic. To better understand this process, we sought to define the stoichiometry of DnaJ in association with the preRC and to identify residues involved in the recognition of this complex. To this end, we have developed a gel filtration-based assay to measure the interaction of DnaJ with the preRC. We find that 3 DnaJ dimers associate per DnaB hexamer in the context of the preRC. Surprisingly, 3 to 4 DnaJ dimers bind λO:DNA in the absence of DnaB suggesting that DnaJ binding to the preRC may be mediated in part through interactions with λO. Mutational analysis reveals a novel substrate binding pocket between DnaJ residues 106-138. An alanine scan across this region identified residues I136 and I138 as critical to DnaJ function. Indeed, mutation of these residues significantly decreases DnaJ:preRC stoichiometry and reduces binding to the preRC as measured by surface plasmon resonance.