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Molecular modeling-based analysis of interactions in the RFC-dependent clamp-loading process

eslovas Venclovas¹, Michael E. Colvin and Michael P. Thelen

Computational and Systems Biology Division, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94550, USA

Reprint requests to: eslovas Venclovas, Computational and Systems Biology Division, Lawrence Livermore National Laboratory, L-448, P.O. Box 808, Livermore, CA 94550, USA; e-mail: venclovas{at}llnl.gov; fax: (925) 422-2282.

Replication and related processes in eukaryotic cells require replication factor C (RFC) to load a molecular clamp for DNA polymerase in an ATP-driven process, involving multiple molecular interactions. The detailed understanding of this mechanism is hindered by the lack of data regarding structure, mutual arrangement, and dynamics of the players involved. In this study, we analyzed interactions that take place during loading onto DNA of either the PCNA clamp or the Rad9-Rad1-Hus1 checkpoint complex, using computationally derived molecular models. Combining the modeled structures for each RFC subunit with known structural, biochemical, and genetic data, we propose detailed models of how two of the RFC subunits, RFC1 and RFC3, interact with the C-terminal regions of PCNA. RFC1 is predicted to bind PCNA similarly to the p21-PCNA interaction, while the RFC3-PCNA binding is proposed to be similar to the E. coli -ß interaction. Additional sequence and structure analysis, supported by experimental data, suggests that RFC5 might be the third clamp loader subunit to bind the equivalent PCNA region. We discuss functional implications stemming from the proposed model of the RFC1-PCNA interaction and compare putative clamp-interacting regions in RFC1 and its paralogs, Rad17 and Ctf18. Based on the individual intermolecular interactions, we propose RFC and PCNA arrangement that places three RFC subunits in association with each of the three C-terminal regions in PCNA. The two other RFC subunits are positioned at the two PCNA interfaces, with the third PCNA interface left unobstructed. In addition, we map interactions at the level of individual subunits between the alternative clamp loader/clamp system, Rad17-RFC_2–5/Rad9-Rad1-Hus1. The proposed models of interaction between two clamp/clamp loader pairs provide both structural framework for interpretation of existing experimental data and a number of specific findings that can be subjected to direct experimental testing.

Keywords: Comparative modeling; AAA+ proteins; DNA replication; clamp loader; pentameric complex; Rad17; Ctf18; 9-1-1 checkpoint complex

Abbreviations: AAA+, ATPases associated with various cellular activities • PCNA, proliferating cell nuclear antigen • RFC, replication factor C • RFCL, archaeal RFC large subunit • RFCS, archaeal RFC small subunit • 9-1-1, Rad9-Hus1-Rad1 checkpoint protein complex

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