TY - JOUR TI - Stepwise assembly of the human replicative polymerase holoenzyme AU - Hedglin, Mark AU - Perumal, Senthil K AU - Hu, Zhenxin AU - Benkovic, Stephen A2 - Kuriyan, John VL - 2 PY - 2013 DA - 2013/04/02 SP - e00278 C1 - eLife 2013;2:e00278 DO - 10.7554/eLife.00278 UR - https://doi.org/10.7554/eLife.00278 AB - In most organisms, clamp loaders catalyze both the loading of sliding clamps onto DNA and their removal. How these opposing activities are regulated during assembly of the DNA polymerase holoenzyme remains unknown. By utilizing FRET to monitor protein-DNA interactions, we examined assembly of the human holoenzyme. The results indicate that assembly proceeds in a stepwise manner. The clamp loader (RFC) loads a sliding clamp (PCNA) onto a primer/template junction but remains transiently bound to the DNA. Unable to slide away, PCNA re-engages with RFC and is unloaded. In the presence of polymerase (polĪ“), loaded PCNA is captured from DNA-bound RFC which subsequently dissociates, leaving behind the holoenzyme. These studies suggest that the unloading activity of RFC maximizes the utilization of PCNA by inhibiting the build-up of free PCNA on DNA in the absence of polymerase and recycling limited PCNA to keep up with ongoing replication. KW - DNA polymerase holoenzyme assembly KW - clamp loading KW - clamp unloading KW - clamp loader KW - sliding clamp JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -