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Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitin-conjugating activity and its various biological functions.

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  • 1. Department of Biology, University of Rochester, NY 14627.
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    • Sung P 1
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Proceedings of the National Academy of Sciences of the United States of America, 01 Apr 1990, 87(7):2695-2699
https://doi.org/10.1073/pnas.87.7.2695 PMID: 2157209 PMCID: PMC53757

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Abstract 

The RAD6 gene of Saccharomyces cerevisiae is required for DNA repair, DNA damage-induced mutagenesis, and sporulation. RAD6 protein is a ubiquitin-conjugating enzyme (E2) that has been shown to attach multiple molecules of ubiquitin to histones H2A and H2B. We have now examined whether the E2 activity of RAD6 is involved in its various biological functions. Since the formation of a thioester adduct between E2 and ubiquitin is necessary for E2 activity, the single cysteine residue (Cys-88) present in RAD6 was changed to alanine or valine. The mutant proteins were overproduced in yeast cells and purified to near homogeneity. We show that the rad6 Ala-88 and rad6 Val-88 mutant proteins lack the capacity for thioester formation with ubiquitin and, as a consequence, are totally devoid of any E2 activity. The rad6 Ala-88 and rad6 Val-88 mutations confer a defect in DNA repair, mutagenesis, and sporulation equivalent to that in the rad6 null allele. We suggest that the biological functions of RAD6 require its E2 activity.

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Proc Natl Acad Sci U S A. 1990 Apr; 87(7): 2695–2699.
PMCID: PMC53757
PMID: 2157209

Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitin-conjugating activity and its various biological functions.

P Sung, S Prakash, and L Prakash
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Abstract

The RAD6 gene of Saccharomyces cerevisiae is required for DNA repair, DNA damage-induced mutagenesis, and sporulation. RAD6 protein is a ubiquitin-conjugating enzyme (E2) that has been shown to attach multiple molecules of ubiquitin to histones H2A and H2B. We have now examined whether the E2 activity of RAD6 is involved in its various biological functions. Since the formation of a thioester adduct between E2 and ubiquitin is necessary for E2 activity, the single cysteine residue (Cys-88) present in RAD6 was changed to alanine or valine. The mutant proteins were overproduced in yeast cells and purified to near homogeneity. We show that the rad6 Ala-88 and rad6 Val-88 mutant proteins lack the capacity for thioester formation with ubiquitin and, as a consequence, are totally devoid of any E2 activity. The rad6 Ala-88 and rad6 Val-88 mutations confer a defect in DNA repair, mutagenesis, and sporulation equivalent to that in the rad6 null allele. We suggest that the biological functions of RAD6 require its E2 activity.

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Selected References

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