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

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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.

Original languageEnglish (US)
Pages (from-to)2695-2699
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume87
Issue number7
StatePublished - Apr 1990
Externally publishedYes

Fingerprint

Saccharomyces cerevisiae Proteins
Ubiquitin
Cysteine
Mutant Proteins
Mutagenesis
DNA Repair
Mutation
Ubiquitin-Conjugating Enzymes
Valine
Alanine
Histones
DNA Damage
Saccharomyces cerevisiae
Yeasts
Alleles
Genes
Proteins

Keywords

  • DNA repair
  • Histone ubiquitination
  • Mutagenesis
  • Sporulation

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

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title = "Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitin-conjugating activity and its various biological functions",
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.",
keywords = "DNA repair, Histone ubiquitination, Mutagenesis, Sporulation",
author = "Patrick Sung and Satya Prakash and Louise Prakash",
year = "1990",
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language = "English (US)",
volume = "87",
pages = "2695--2699",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
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TY - JOUR

T1 - Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitin-conjugating activity and its various biological functions

AU - Sung, Patrick

AU - Prakash, Satya

AU - Prakash, Louise

PY - 1990/4

Y1 - 1990/4

N2 - 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.

AB - 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.

KW - DNA repair

KW - Histone ubiquitination

KW - Mutagenesis

KW - Sporulation

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M3 - Article

VL - 87

SP - 2695

EP - 2699

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 7

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