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J. Biol. Chem., Vol. 276, Issue 13, 10168-10177, March 30, 2001

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Human-Saccharomyces cerevisiae Proliferating Cell Nuclear Antigen Hybrids
OLIGOMERIC STRUCTURE AND FUNCTIONAL CHARACTERIZATION USING IN VITRO DNA REPLICATION^*

Ayodele Ola, Shou Waga^§¶, Viola Ellison^§, Bruce Stillman^§, Mark McGurk, Irene M. Leigh, Naushin H. Waseem^**, and Ahmad Waseem

From the  Head and Neck Cancer Research Program, Guys, King's, and St. Thomas' Dental Institute and the ^** Division of Molecular and Medical Genetics, Pediatrics Research Unit, Guy's Campus, King's College London, London SE1 9RT, United Kingdom, the ^§ Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, and the  Imperial Cancer Research Fund Skin Tumor Laboratory, the Royal London Hospital, London E12AT, United Kingdom

The proliferating cell nuclear antigen (PCNA) is a highly conserved protein required for the assembly of the DNA polymerase delta (pol ) holoenzyme. Because PCNAs from Saccharomyces cerevisiae and human do not complement each other using in vitro or in vivo assays, hybrids of the two proteins would help identify region(s) involved in the assembly of the pol  holoenzyme. Two mutants of human PCNA, HU1 (D21E) and HU3 (D120E), and six hybrids of human and S. cerevisiae PCNA, HC1, HC5, CH2, CH3, CH4, and CH5, were prepared by swapping corresponding regions between the two proteins. In solution, all PCNA assembled into trimers, albeit to different extents. These PCNA variants were tested for stimulation of pol  and in vitro replication of M13 and SV40 DNA as well as to stimulate the ATPase activity of replication factor C (RF-C). Our data suggest that in addition to the interdomain connecting loop and C terminus, an additional site in the N terminus is required for pol  interaction. PCNA mutants and hybrids that stimulated pol  and RF-C were deficient in M13 and SV40 DNA replication assays, indicating that PCNA-induced pol  stimulation and RF-C-mediated loading are not sufficient for coordinated DNA synthesis at a replication fork.

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