Disease relevance of RAD6

• Under mild heat stress conditions (37-38 degrees C) rad6 null and rad6-149 mutant cells are unable to grow [1].
• The recombinant yeast RAD6 and CDC34 gene products were expressed in Escherichia coli extracts and purified to apparent homogeneity [2].
• This pattern of Rad6 protein expression/localization is not restricted to breast cancer cell lines, because human breast carcinomas display similar patterns of Rad6 up-regulation and nuclear localization suggesting that deregulation in expression of Rad6 may be an important step in transformation to malignant phenotype [3].
• Metabolic suppressors of trimethoprim and ultraviolet light sensitivities of Saccharomyces cerevisiae rad6 mutants [4].
• Overexpression of Rad6 in ovarian cancer promotes stem cell characteristics and resistance to platinum drugs  [5] .
• Novel small molecule inhibitors of Rad6 have anticancer activity [6].

High impact information on RAD6

• The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood [7].
• We also show that an H2B-K123R mutation perturbs silencing at the telomere, providing functional links between Rad6-mediated H2B (Lys 123) ubiquitination, Set1-mediated H3 (Lys 4) methylation, and transcriptional silencing [8].
• Here we show that the ubiquitin-conjugating enzyme Rad6 (Ubc2) mediates methylation of histone H3 at lysine 4 (Lys 4) through ubiquitination of H2B at Lys 123 in yeast (Saccharomyces cerevisiae) [8].
• The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme [9].
• The RAD6 gene of the yeast Saccharomyces cerevisiae is required for a variety of cellular functions including DNA repair [9].

Biological context of RAD6

• In addition to the tail, the cell cycle function exhibited by the chimera and CDC34 is probably dependent on a conserved region of the catalytic domain that is shared by both RAD6 and CDC34 [10].
• Saccharomyces cerevisiae MGS1 is essential in strains deficient in the RAD6-dependent DNA damage tolerance pathway [11].
• Despite this similarity, the CDC34 catalytic domain cannot substitute for the DNA repair and growth functions of the RAD6 catalytic domain, indicating that although these domains are structurally related, sufficient differences exist to maintain their functional individuality [10].
• These findings suggest that Mgs1 is essential for preventing genome instability caused by replication fork arrest in cells deficient in the RAD6 pathway and may modulate replication fork movement catalyzed by yeast polymerase delta [11].
• The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme required for postreplicational repair of UV-damaged DNA and for damage-induced mutagenesis [12].

Anatomical context of RAD6

• A rabbit reticulocyte ubiquitin carrier protein that supports ubiquitin-dependent proteolysis (E214k) is homologous to the yeast DNA repair gene RAD6 [13].
• Since RAD6 and its mammalian homologs can ubiquitinate histones in vitro, we have investigated the pattern of histone ubiquitination in mouse testis [14].
• This physical association of Rad6 with centrosomes is maintained throughout the interphase and mitotic phases of the cell cycle [3].

Associations of RAD6 with chemical compounds

• The conserved proline residue of RAD6 and CDC34 is part of a turn motif common to all ubiquitin-conjugating enzymes [15].
• Strains carrying mutations in either the RAD3, RAD6 or RAD52 genes were treated with increasing concentrations of 4-nitroquinoline-1-oxide (NQO) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the DDR transcript levels were determined by Northern hybridization analysis [16].
• Our current model suggests that the Rad6-Rad18 complex targets Poleta at DNA gaps that result from the MMR-mediated excision of adenine mispaired with 8-oxoG, allowing error-free dCMP incorporation opposite to this lesion [17].
• We have investigated this mechanism in Saccharomyces cerevisiae, in which it is the major component of the RAD6/RAD18 pathway, by transforming an isogenic set of rad1Delta excision-defective strains with plasmids that carry a single thymine-thymine pyrimidine (6-4) pyrimidinone photoadduct in each strand at staggered positions 28 base pairs apart [18].
• Interaction between RAD6 and RAD52 genes was epistatic for low bleomycin concentrations [19].

Physical interactions of RAD6

• In its role in N end-dependent protein degradation, Rad6 interacts with the UBR1-encoded ubiquitin protein ligase (E3) enzyme [12].
• Here, we show that RAD6 forms a specific complex with the product of the DNA repair gene RAD18 [20].
• One of these patches corresponds to a binding site for both HECT and RING domain proteins, suggesting that a single substitution in the catalytic domain of RAD6 confers upon it the ability to interact with multiple ubiquitin protein ligases (E3s) [21].
• We also show that Paf1 complex is required for the interaction of Rad6 and COMPASS with RNA polymerase II [22].

Enzymatic interactions of RAD6

• RAD6 and E2(20k) exhibit marked specificity for the conjugation of core histones and catalyze the processive ligation of up to three ubiquitin moieties directly to such model substrates [2].

Regulatory relationships of RAD6

• Overexpression of WSC2 reverses the heat-induced cell cycle arrest of rad6-149 but not of rad6 null mutants [1].
• srs2 was isolated during a screen for mutants that could suppress the UV-sensitive phenotype of rad6 and rad18 cells [23].

Other interactions of RAD6

• Furthermore, the disruption of the BRE1 as well as substitution of the ubiquitylation site of histone H2B also reduces some DSB formation similar to the rad6 [24].
• The rad6 and rad18 mutants are defective in both pathways, and the rev3 mutant affects only the mutagenesis pathway, but a yeast gene that is involved only in error-free postreplication repair has not been reported [25].
• We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Polzeta, error-free translesion synthesis by Poleta, and postreplication repair of discontinuities by a Rad5-dependent pathway [26].
• This is most clear for the repair of adducts on the transcribed strand, where an absolute requirement for Rad6 and Rad18 was seen [27].
• Surprisingly, the E2(14k)) sequence is markedly more similar to Saccharomyces cerevisiae RAD6 (69% identity) than to its proposed homologs UBC4/UBC5 (38% identity) [13].

Analytical, diagnostic and therapeutic context of RAD6

• Dissection of the functions of the Saccharomyces cerevisiae RAD6 postreplicative repair group in mutagenesis and UV sensitivity [28].
• Sequence analysis demonstrated an identity of 69% in the primary sequence of human E2(Mr = 17,000) and the protein encoded by the yeast DNA repair gene RAD6, which was recently shown to be an E2 species in yeast [29].
• Molecular cloning via complementation of the pso8 mutant's sensitivity phenotype and genetic studies revealed that pso8 is allelic to RAD6 [30].
• Western blot analysis was used to probe the relationships between the multiple ubiquitin carrier proteins (E2 s) of rabbit reticulocytes and the 20-kDa E2 encoded by the RAD6 gene of the yeast S. cerevisiae [31].
• Chromatin immunoprecipitation experiments indicated that both Rad6 and Bre1 are recruited to a promoter [32].

References