FIG. 6.

Recombinant RPA/SSB, RFC, PCNA, and Pol δ replicate primed M13 DNA. (A) SDS-PAGE analyses of the purified human recombinant RPA, RFC, PCNA, and Pol δ preparations used in replication and protein-protein interaction assays (see Materials and Methods for details). The positions of the relevant purified proteins or subunits are indicated on the right of each gel, and the positions of a standard set of molecular mass markers run in the first lane of each gel are shown on the left. The gels were stained with Coomassie brilliant blue. (B) Autoradiograph of an M13 singly primed extension assay analyzed by alkaline agarose gel electrophoresis. Singly primed M13 DNA (7.3 kb) was incubated with deoxynucleoside triphosphates, including [³²P]dATP and ATP, in the presence (+) or absence (−) of titrated amounts of purified recombinant RFC, PCNA, Pol δ, and RPA or SSB. For reactions including RPA or E. coli SSB, the amounts added were 0.5 or 1.5 μg, respectively, and elsewhere + represents addition of 1.5 μg of either protein. RFC titrations were 5 and 25 ng, and elsewhere + represents 125 ng of protein added. PCNA titrations contained 10 or 25 ng, and elsewhere + indicates 100 ng of protein added. For Pol δ titrations, 5 or 25 ng was added to the assay, and elsewhere + represents 125 ng of protein added. The first lane contains a ³²P-labeled, HindIII-digested λ phage DNA marker (M), while the arrows on the left indicate the sizes of individual fragments. The amount of dAMP (in picomoles) incorporated into each replication product is indicated in the histogram at the bottom.