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. 2007 Nov 5;36(1):67–75. doi: 10.1093/nar/gkm943

Figure 6.

Figure 6.

PCNA binding motif-dependent recruitment of MMR proteins to replicating DNA Panel (A) shows an outline of the experimental approach. After a 20 min incubation, to set up active DNA replication/repair complexes, either p21C or the p21C mutant is added. After 10 min, the complexes are subjected to gel filtration and immunoblotted as above. Panel (B) shows that at the 20 min time point that recruitment of RPA, PCNA and MSH2 to the replicating DNA can be readily detected via immunoblotting. In Panel (C) either GST-p21C, GST-p21C mutant or GST alone (34 µg/ml of each) were added to the replication reaction at 20 min. DNA incorporation was evaluated as described in Materials and Methods section and plotted as counts per minute (C.P.M.) incorporated at each of the indicated times. p21C (34 µg/ml; Panel D), or p21Cmut (34 µg/ml; Panel E), was added at 20 min during the SV40 DNA replication reaction. The reaction was further incubated for 10 min and then subjected to gel filtration. Precipitated protein fractions were analyzed by immunoblotting for MSH2, RPA70 and PCNA as above. As a control for antibody recognition, lanes marked CE contained 5 µl of cell extract representing 7% of the proteins in the entire reaction.