Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by theEscherichia coliDNA Polymerase III Holoenzyme

摘要: The DnaX complex (DnaX3δδ′χψ) within the Escherichia coli DNA polymerase III holoenzyme serves to load dimeric sliding clamp processivity factor, β2, onto DNA. contains three subunits, which occur in two forms: τ and shorter γ, produced by translational frameshifting. Ten forms of E. containing all possible combinations wild-type or a Walker A motif K51E variant γ have been reconstituted rigorously purified. complexes subunits do not bind ATP. Comparison their ability support formation initiation complexes, as measured processive replication holoenzyme, indicates minimal requirement for one ATP-binding subunit. mutant synthesis at about two-thirds level counterparts. β2 binding (determined functionally) is diminished 12–30-fold suggesting that multiple ATPs must be bound place into conformation with maximal affinity β2. activity can restored increased concentrations In contrast, severe defects ATP hydrolysis are observed upon introduction single Thus, binding, hydrolysis, form tightly coupled. These results suggest although likely enhances loading, it absolutely required mechanistic sense functional complexes.