tailieunhanh - Báo cáo khoa học: Inactivating pentapeptide insertions in the fission yeast replication factor C subunit Rfc2 cluster near the ATP-binding site and arginine finger motif

Replication factor C (RFC) plays a key role in eukaryotic chromosome replication by acting as a loading factor for the essential sliding clamp and polymerase processivity factor, proliferating cell nuclear antigen (PCNA). RFC is a pentamer comprising a large subunit, Rfc1, and four small subunits, Rfc2–Rfc5. | ỊFEBS Journal Inactivating pentapeptide insertions in the fission yeast replication factor C subunit Rfc2 cluster near the ATP-binding site and arginine finger motif Fiona C. Gray 1 2 Kathryn A. Whitehead1 and Stuart A. MacNeill1 2 f 1 Wellcome Trust Centre for CellBiology University of Edinburgh UK 2 Department of Biology University of Copenhagen Denmark Keywords AAA protein clamp loader DNA replication fission yeast replication factor C Correspondence S. MacNeill Centre for Biomolecular Sciences University of St Andrews North Haugh St Andrews KY16 9ST UK Fax 44 01334 462595 Tel 44 01334 467268 E-mail Website http macneill Present addresses Department of Chemistry and Materials Manchester Metropolitan University UK fCentre for Biomolecular Sciences University of St Andrews UK Received 20 March 2009 revised 24 June 2009 accepted 26 June 2009 Replication factor C RFC plays a key role in eukaryotic chromosome replication by acting as a loading factor for the essential sliding clamp and polymerase processivity factor proliferating cell nuclear antigen PCNA . RFC is a pentamer comprising a large subunit Rfc1 and four small subunits Rfc2-Rfc5. Each RFC subunit is a member of the AAA family of ATPase and ATPase-like proteins and the loading of PCNA onto double-stranded DNA is an ATP-dependent process. Here we describe the properties of a collection of 38 mutant forms of the Rfc2 protein generated by pentapeptide-scanning mutagenesis of the fission yeast rfc2 gene. Each insertion was tested for its ability to support growth in fission yeast rfc2D cells lacking endogenous Rfc2 protein and the location of each insertion was mapped onto the 3D structure of budding yeast Rfc2. This analysis revealed that the majority of the inactivating mutations mapped in or adjacent to ATP sites C and D in Rfc2 arginine finger and P-loop respectively or to the five-stranded b sheet at the heart of the Rfc2 protein. By contrast .