ATP Hydrolysis Catalyzed by Human Replication Factor C Requires Participation of Multiple Subunits

• Published in: Proceedings of the National Academy of Sciences - PNAS 1998-09, Vol.95 (20), p.11607-11612
• Main Authors: Cai, Jinsong, Yao, Nina, Gibbs, Emma, Finkelstein, Jeff, Phillips, Barbara, O'Donnell, Michael, Hurwitz, Jerard
• Format: Article
• Language: English
• Subjects: Adenosine triphosphatases; Adenosine Triphosphate - metabolism; Amino Acid Sequence; Amino acids; Baculoviridae; Binding sites; Binding Sites - genetics; Biochemistry; Biological Sciences; Deoxyribonucleic acid; DNA; DNA - biosynthesis; DNA Replication; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Genetic mutation; Homeodomain Proteins; Humans; Hydrolysis; In Vitro Techniques; Insect genetics; Insect viruses; Minor Histocompatibility Antigens; Models, Biological; Molecules; Mutagenesis, Site-Directed; Point Mutation; Proliferating Cell Nuclear Antigen - metabolism; Protein Conformation; Proteins; Proto-Oncogene Proteins c-bcl-2; Replication Protein C; Repressor Proteins; Saccharomyces cerevisiae Proteins
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.20.11607

Human replication factor C (hRFC) is a five-subunit protein complex (p140, p40, p38, p37, and p36) that acts to catalytically load proliferating cell nuclear antigen onto DNA, where it recruits DNA polymerase δ or ε to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomplex of hRFC consisting of three subunits (p40, p37, and p36) contained DNA-dependent ATPase activity. However, it is not clear which subunit(s) hydrolyzes ATP, as all five subunits include potential ATP binding sites. In this report, we introduced point mutations in the putative ATP-binding sequences of each hRFC subunit and examined the properties of the resulting mutant hRFC complex and the ATPase activity of the hRFC or the $\text{p40}· \text{p37}· \text{p36}$ complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduced replication activity of the hRFC complex and the ATPase activity of the hRFC or the $\text{p40}· \text{p37}· \text{p36}$ complex. A mutation in the ATP binding site of the p38 subunit did not alter the replication activity of hRFC. These findings indicate that the replication activity of hRFC is dependent on efficient ATP hydrolysis contributed to by the action of four hRFC subunits.