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Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis

Abstract

The rapid growth in ubiquitin biology requires facile chemical approaches for protein ubiquitylation that can overcome the common problem of low yield faced by the enzymatic reaction catalyzed by ubiquitin ligases. We report a chemical approach for monoubiquitylation and SUMOylation of PCNA through disulfide exchange and intein chemistry. We used the chemically ubiquitylated and SUMOylated PCNAs in studying translesion DNA synthesis and revealed a surprising degree of flexibility of the ubiquitin modification.

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Figure 1: Chemical ubiquitylation of PCNA.
Figure 2: The function of chemically ubiquitylated PCNA in the polymerase exchange assay and the pulldown assay.

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Acknowledgements

We thank S. Prakash (University of Texas Medical Branch) for the generous gift of yeast Polδ. We also thank C. Thorpe for helpful discussions. This work was supported in part by a grant from the University of Delaware Research Foundation to Z.Z. (UDRF08000654). L.H. acknowledges funding from the Hungarian Science Foundation (OTKA 77495-TAMOP-4.2.2/08/1).

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Contributions

J.C., Y.A. and Z.Z. designed research; J.C., Y.A. and J.W. performed research; L.H. contributed new reagents; J.C., Y.A. and Z.Z. analyzed data; and Z.Z. wrote the paper.

Corresponding author

Correspondence to Zhihao Zhuang.

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The authors declare no competing financial interests.

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Supplementary Methods and Supplementary Figures 1–19 (PDF 998 kb)

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Chen, J., Ai, Y., Wang, J. et al. Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis. Nat Chem Biol 6, 270–272 (2010). https://doi.org/10.1038/nchembio.316

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