The hexameric AAA+ ring of Escherichia. coli ClpX, an ATP-dependent protein unfolding and translocation machine, functions with the ClpP peptidase to degrade target substrates. For efficient function, ClpX subunits must switch between nucleotide-loadable (L) and nucleotide-unloadable (U) conformations, but the roles of switching are uncertain. Moreover, it is controversial whether working AAA+ ring enzymes assume symmetric or asymmetric conformations. Here, we show that a covalent ClpX ring with one subunit locked in the U conformation catalyzes robust ATP-hydrolysis, with each unlocked subunit able to bind and hydrolyze ATP, albeit with highly asymmetric position-specific affinities. Preventing U⇔L interconversion in one subunit alters the cooperativity of ATP hydrolysis and reduces the efficiency of substrate binding, unfolding, and degradation, showing that conformational switching enhances multiple aspects of wild-type ClpX function. These results support an asymmetric and probabilistic model of AAA+ ring activity.

Enzymatic processing of DNA underlies all DNA repair, yet inappropriate DNA processing must be avoided. In vertebrates, double-strand breaks are repaired predominantly by non-homologous end-joining (NHEJ), which directly ligates DNA ends. NHEJ has the potential to be highly mutagenic because it employs DNA polymerases, nucleases, and other enzymes that modify incompatible DNA ends to allow their ligation. Using a biochemical system that recapitulates key features of cellular NHEJ, we show that endprocessing requires formation of a "short-range synaptic complex" in which DNA ends are closely aligned in a ligation-competent state. Furthermore, single-molecule imaging directly demonstrates that processing occurs within the short-range complex. This confinement of end processing to a ligation-competent complex ensures that DNA ends undergo ligation as soon as they become compatible, thereby minimizing mutagenesis.Our results illustrate how the coordination of enzymatic catalysis with higher-order structural organization of substrate maximizes the fidelity of DNA repair.