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The DNA repair protein Rad23 is a negative regulator of multi-ubiquitin chain assembly

Nature Cell Biology volume 2, pages 601–608 (2000)Cite this article

Abstract

Rad23 is a nucleotide-excision repair protein with a previously unknown biochemical function. We determined that yeast and human Rad23 inhibited multi-ubiquitin (Ub) chain formation and the degradation of proteolytic substrates. Significantly, Rad23 could be co-precipitated with a substrate that contained a short multi-Ub chain. The UV sensitivity of rad23Δ was reduced in mutants lacking the E2 enzyme Ubc4, or the multi-Ub chain-promoting factor Ufd2. These studies suggest that the stability of proteolytic substrates is governed by the competing action of multi-Ub chain-promoting and chain-inhibiting factors. The stabilization of DNA repair and stress factors could represent an important biological function of Rad23.

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Figure 1: Rad23 inhibits multi-Ub chain assembly.
Figure 2: Ubc2 assembles a multi-Ub chain on histone H2B.
Figure 3: Rad23 does not form a thioester bond with Ub, and does not prevent E1 and E2 from forming this intermediate.
Figure 4: Domain structure and conservation of Rad23 function.
Figure 5: Rad23 can interfere with the degradation of proteolytic substrates in vivo.
Figure 6: A role for proteolysis in nucleotide-excision repair.

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Acknowledgements

We thank I. Vega for recombinant Rad23 protein and for constructing a plasmid that expressed ΔUbLrad23. We also thank J. Huibregtse for providing reagents to examine the effect of Rad23 on Rsp5-mediated ubiquitination in rabbit reticulocyte lysates, D. Stern for the GST–Spk1 construct and C. Pickart for plasmids pGEX-E2-25K and pJY2. M. Ellison, M. Hochstrasser and D. Gietz are thanked for providing plasmids and strains. K. Sweder is thanked for assistance with the NIH Scan densitometry analysis. D.L. was supported by a pre-doctoral fellowship from the American Heart Association (9810001T). C.S. was supported by Postdoctoral Fellowships from the Robert Wood Johnson Foundation and New Jersey Commission on Cancer Research (98-2005-CCR-00). These studies were supported by grants to K.M. from the National Institutes of Health (GM-RO1-52058) and the Cancer Institute of New Jersey (561735). K.M. is a member of the Cancer Institute of New Jersey.

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Author notes

  1. Prasad Tongaonkar

    Present address: Department of Biological Chemistry, University of California, Irvine, California, 92967, USA

  2. Cherylene Schauber

    Present address: Department of Biological Sciences, Stanford University, Stanford, California, 94305, USA

Authors and Affiliations

  1. Department of Biochemistry, Robert Wood Johnson Medical School-UMDNJ, 675 Hoes Lane, Piscataway, 08854, New Jersey, USA

    Tatiana G. Ortolan, Prasad Tongaonkar, David Lambertson, Li Chen, Cherylene Schauber & Kiran Madura

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Correspondence to Kiran Madura.

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Ortolan, T., Tongaonkar, P., Lambertson, D. et al. The DNA repair protein Rad23 is a negative regulator of multi-ubiquitin chain assembly. Nat Cell Biol 2, 601–608 (2000). https://doi.org/10.1038/35023547

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