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 (PCNA) are processivity factors for eukaryotic DNA polymerases delta and epsilon. RFC contains multiple activities, including its ability to recognize and bind to a DNA primer end and load the ring-shaped PCNA onto DNA in an ATP-dependent reaction. PCNA then tethers the polymerase to the template allowing processive DNA chain elongation. Human RFC consists of five distinct subunits (p140, p40, p38, p37, and p36), and RFC activity can be reconstituted from the five cloned gene products. To characterize the role of the large subunit p140 in the function of the RFC complex, deletion mutants were created that defined a region within the p140 C terminus required for complex formation with the four small subunits. Deletion of the p140 N-terminal half, including the DNA ligase homology domain, resulted in the formation of an RFC complex with enhanced activity in replication and PCNA loading. Deletion of additional N-terminal amino acids, including those constituting the RFC homology box II that is conserved among all five RFC subunits, disrupted RFC replication function. DNA primer end recognition and PCNA binding activities, located in the p140 C-terminal half, were unaffected in this mutant, but PCNA loading was abolished.
Uhlmann
F
F
Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Cai
J
J
Gibbs
E
E
O'Donnell
M
M
Hurwitz
J
J
D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004270:DNA, Circular / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D018398:Homeodomain Proteins; D006801:Humans; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011487:Protein Conformation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
C096816:BCL2-related protein A1; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; C497562:RFC3 protein, human; C497563:RFC4 protein, human; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D051818:Replication Protein C