DELETION ANALYSIS OF THE LARGE SUBUNIT P140 IN HUMAN REPLICATION FACTOR-C REVEALS REGIONS REQUIRED FOR COMPLEX-FORMATION AND REPLICATION ACTIVITIES

Replication factor C (RFC) and proliferating cell nuclear antigen (PCN A) are processivity factors for eukaryotic DNA polymerases delta and e psilon. RFC contains multiple activities, including its ability to rec ognize and bind to a DNA printer end and load the ring-shaped PCNA ont o DNA in an ATP-dependent reaction. PCNA then tethers the polymerase t o the template allowing processive DNA chain elongation. Human RFC con sists of five distinct subunits (P140, p40, p38, p37, and p36), and RF C activity can be reconstituted from the five cloned gene products. To characterize the role of the large subunit p140 in the function of th e RFC complex, deletion mutants were created that defined a region wit hin the p140 C terminus required for complex formation with the four s mall subunits. Deletion of the p140 N-terminal half, including the DNA ligase homology domain, resulted in the formation of all RFC complex with enhanced activity in replication and PCNA loading. Deletion of ad ditional N-terminal amino acids, including those constituting the RFC homology box II that is conserved among all five RFC subunits, disrupt ed RFC replication function. DNA primer end recognition and PCNA bindi ng activities, located in the p140 C-terminal half, were unaffected in this mutant, but PCNA loading was abolished.