Fig. 5.

Exo1-independent mismatch repair in a 2-stage reaction. Single-stage reactions (gray bars) contained 5′ G–T heteroduplex DNA, ATP, the 4 dNTPs, and the protein components as indicated on the abscissa (see Materials and Methods; D699N indicates substitution of MutLα by MutLαD699N). Repair was scored by cleavage with HindIII and ClaI (Fig. 1). No detectable repair was observed in the presence of only polymerase δ or polymerase δ plus PCNA. In 2-stage reactions (black bars), the 5′ G–T heteroduplex was preincised by 30-min incubation with MutSα, MutLα, RFC, PCNA, RPA, the 4 dNTPs, and ATP, followed by deproteinization (see Materials and Methods). Isolated DNA was then incubated for 30 min with ATP, the 4 dNTPs, and the indicated protein components. Repair was scored with HindIII and ClaI as above.