The replication factor C clamp loader requires arginine finger sensors to drive DNA binding and proliferating cell nuclear antigen loading.
Replication factor C (RFC) is an AAA+ heteropentamer that couples the energy of ATP binding and hydrolysis to the loading of the DNA polymerase processivity clamp, proliferating cell nuclear antigen (PCNA), onto DNA. RFC consists of five subunits in a spiral arrangement (RFC-A, -B, -C, -D, and -E, corresponding to subunits RFC1, RFC4, RFC3, RFC2, and RFC5, respectively). The RFC subunits are AAA+ family proteins and the complex contains four ATP sites (sites A, B, C, and D) located at subunit interfaces. In each ATP site, an arginine residue from one subunit is located near the gamma-phosphate of ATP bound in the adjacent subunit. These arginines act as "arginine fingers" that can potentially perform two functions: sensing that ATP is bound and catalyzing ATP hydrolysis. In this study, the arginine fingers in RFC were mutated to examine the steps in the PCNA loading mechanism that occur after RFC binds ATP. This report finds that the ATP sites of RFC function in distinct steps during loading of PCNA onto DNA. ATP binding to RFC powers recruitment and opening of PCNA and activates a gamma-phosphate sensor in ATP site C that promotes DNA association. ATP hydrolysis in site D is uniquely stimulated by PCNA, and we propose that this event is coupled to PCNA closure around DNA, which starts an ordered hydrolysis around the ring. PCNA closure severs contact to RFC subunits D and E (RFC2 and RFC5), and the gamma-phosphate sensor of ATP site C is switched off, resulting in low affinity of RFC for DNA and ejection of RFC from the site of PCNA loading.
Johnson
Aaron
A
Laboratory of DNA Replication, Howard Hughes Medical Institute and Rockefeller University, New York, New York 10021, USA.
Yao
Nina Y
NY
Bowman
Gregory D
GD
Kuriyan
John
J
O'Donnell
Mike
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D001120:Arginine; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051818:Replication Protein C / Q000737:chemistry* / Q000378:metabolism*
D018809:Proliferating Cell Nuclear Antigen; D000255:Adenosine Triphosphate; D004247:DNA; D001120:Arginine; D051818:Replication Protein C