ATP-dependent structural change of the eukaryotic clamp-loader protein, replication factor C

The eukaryotic DNA sliding clamp that keeps DNA polymerase engaged at a rep lication fork, called proliferating cell nuclear antigen (PCNA), is loaded onto the 3' ends of primer DNA through its interaction with a heteropentame ric protein complex called replication factor C (RFC). The ATPase activity of RFC is necessary for formation of a functional PCNA clamp. In the presen t study, the sensitivity of RFC to partial proteolysis is used to show that addition of ATP, ATP gammaS, or ADP induces different structural changes i n RFC. Direct observation by electron microscopy reveals that RFC has a clo sed two-finger structure called the U form in the absence of ATP. This is c onverted into a more open C form on addition of ATP. in contrast, the struc tural changes induced by ATP gammaS or ADP are limited. These results sugge st that RFC adapts on opened configuration intermediately after ATP hydroly sis. We further observe that PCNA is held between the two fingers of RFC an d propose that the RFC structure change we observe during ATP hydrolysis ca uses the attached PCNA to form its active ring-like clamp on DNA.