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RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO

Abstract

The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood. Two principal elements of this pathway are the ubiquitin-conjugating enzymes RAD6 and the MMS2–UBC13 heterodimer, which are recruited to chromatin by the RING-finger proteins RAD18 and RAD5, respectively. Here we show that UBC9, a small ubiquitin-related modifier (SUMO)-conjugating enzyme, is also affiliated with this pathway and that proliferating cell nuclear antigen (PCNA)—a DNA-polymerase sliding clamp involved in DNA synthesis and repair—is a substrate. PCNA is mono-ubiquitinated through RAD6 and RAD18, modified by lysine-63-linked multi-ubiquitination—which additionally requires MMS2, UBC13 and RAD5—and is conjugated to SUMO by UBC9. All three modifications affect the same lysine residue of PCNA, suggesting that they label PCNA for alternative functions. We demonstrate that these modifications differentially affect resistance to DNA damage, and that damage-induced PCNA ubiquitination is elementary for DNA repair and occurs at the same conserved residue in yeast and humans.

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Figure 1: PCNA is modified by SUMO in S. cerevisiae.
Figure 2: Regulation of PCNA SUMO modification and ubiquitination by DNA damage and link to the RAD6 pathway.
Figure 3: Role of PCNA modifications in DNA repair.
Figure 4: Human PCNA is ubiquitinated at the conserved K164 residue on DNA damage.
Figure 5: Model for ubiquitination and SUMO modification of PCNA.

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Acknowledgements

We thank U. Cramer for technical assistance and S. Müller and M. Knop for experimental advice and discussions. We also thank P. Burgers, D. Finley, M. Hochstrasser, M. Knop, S. Müller and B. Stillman for plasmids and strains. S.J. is supported by the Max Planck Society, Deutsche Forschungsgemeinschaft and Fonds der chemischen Industrie.

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Correspondence to Stefan Jentsch.

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Hoege, C., Pfander, B., Moldovan, GL. et al. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002). https://doi.org/10.1038/nature00991

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