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A functional analysis of PCNA-binding peptides derived from protein sequence, interaction screening and rational design

Oncogene volume 25, pages 2850–2859 (2006)Cite this article

Abstract

Proliferating cell nuclear antigen (PCNA) has no intrinsic enzymatic function, but functions as a sliding platform to mediate protein interactions with the DNA strand. Many proteins interact with PCNA through a small conserved motif with consensus QxxLxxFF. This work uses Schizosaccharomyces pombe and human cells to analyse the function of PCNA-binding peptides. Interacting peptides were identified using two-hybrid screening; one (pep102) binds directly to a physiologically relevant site on PCNA. The EGFP-pep102 overexpression phenotype is consistent with competitive blocking of PCNA–protein interactions. Various PCNA-binding peptides were all shown to inhibit PCNA function by competitive binding in both human and S. pombe cells as EGFP fusion proteins. The action of a p21(WAF1/Cip1)-derived peptide was complicated by the presence of additional functional domains and possible post-translational modification. The activity of pep102 was hampered by low expression in both model systems. The peptide derived from rational design (con1) was stable, highly active in inhibiting PCNA function both S. pombe and human cells and showed a high affinity for PCNA both in vitro and in vivo. These results validate the use of functional screening in yeast to identify peptide aptamers that are functional in mammalian cells; such aptamers provide excellent leads for small molecule antiproliferative therapies.

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Acknowledgements

I thank Tony Carr for the nmt-EGFP plasmid and Daniella Zheleva for purified human PCNA. I also thank Sylvie Tournier for plasmids and helpful discussions and Alison Sparks for her help with FACS analysis. I thank all my colleagues for their help and support, especially David Lane and the members of his laboratory. This work was supported by the Association for International Cancer Research and the Cancer Research UK.

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  1. Department of Surgery and Molecular Oncology, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK

    E Warbrick

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Warbrick, E. A functional analysis of PCNA-binding peptides derived from protein sequence, interaction screening and rational design. Oncogene 25, 2850–2859 (2006). https://doi.org/10.1038/sj.onc.1209320

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