After completion of an Okazaki fragment, β clamps are releas…
After completion of an Okazaki fragment, β clamps are released from Pol III and recycled. In order to recycle the clamps, the clamps must be unloaded from the DNA. What unloads the clamps, or do the clamps spontaneously unload themselves after completion of an Okazaki fragment? To examine this question, the following experiment was performed by the O’Donnell lab. A substrate was prepared by loading 32P-labeled β clamp onto a circular nicked double-stranded DNA, then the substrate was incubated for 0-1500 seconds with either purified Pol III (panel C), γ complex (panel B), purified δ subunit of the clamp loader (panel D), or substrate alone (panel A). Incubation reactions were then size-fractionated on an agarose gel, and the gels were subjected to autoradiography on X-ray film. In panel D, δ subunits were incubated with substrate for 5 minutes with increasing amounts of δ subunit, then fractionated on an agarose gel, and autoradiographed as in the other experiments. The upper band in the gels represents the β clamp complexed with the DNA substrate, the lower band represents free clamp. Based on the results below, which of the components assayed has clamp unloading activity?
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