Requirement of ELC1 for RNA polymerase II polyubiquitylation and degradation in response to DNA damage in Saccharomyces cerevisiae

Abstract

Treatment of Saccharomyces cerevisiae and human cells with DNA-damaging agents such as UV light or 4-nitroquinoline-1-oxide induces polyubiquitylation of the largest RNA polymerase II (Pol II) subunit, Rpb1, which results in rapid Pol II degradation by the proteasome. Here we identify a novel role for the yeast Elc1 protein in mediating Pol II polyubiquitylation and degradation in DNA-damaged yeast cells and propose the involvement of a ubiquitin ligase, of which Elc1 is a component, in this process. In addition, we present genetic evidence for a possible involvement of Elc1 in Rad7-Rad16-dependent nucleotide excision repair (NER) of lesions from the nontranscribed regions of the genome and suggest a role for Elc1 in increasing the proficiency of repair of nontranscribed DNA, where as a component of the Rad7-Rad16-Elc1 ubiquitin ligase, it would promote the efficient turnover of the NER ensemble from the lesion site in a Rad23-19S proteasomal complex-dependent reaction.

FIG. 1.

The elc1Δ mutation enhances the UV sensitivity of deletions in genes of the RAD6 and RAD52 epistasis groups but has no effect on the UV sensitivity of the rad14Δ mutation defective in NER. YPD plates containing 5 μl of serial 10-fold dilutions of exponentially growing yeast cells were UV irradiated and incubated in the dark at 30°C.

FIG. 2.

Epistatic interaction of the elc1Δ mutation with the rad7Δ, rad16Δ, and rad23Δ mutations. YPD plates containing 5 μl of serial 10-fold dilutions of exponentially growing yeast cells were UV irradiated and incubated in the dark at 30°C.

FIG. 3.

The elc1Δ mutation enhances the UV sensitivity of the rad26Δ mutation. YPD plates containing 5 μl of serial 10-fold dilutions of exponentially growing yeast cells were UV irradiated and incubated in the dark at 30°C.

FIG. 4.

Rpb1 is not degraded in UV- or 4-NQO-treated elc1Δ cells. (A) Rpb1 levels in wild-type, elc1Δ, and NER-defective mutant strains following UV treatment. Yeast cells were UV irradiated as described in Materials and Methods, and cell extracts were prepared at the indicated times after UV treatment. Rpb1 and the loading control PGK1 were detected by SDS-PAGE, followed by immunoblotting with MAb 8WG16 and MAb PGK1, respectively. (B) Rpb1 levels in wild-type and elc1Δ strains following treatment with 4-NQO. 4-NQO was added to liquid cultures of log-phase yeast cells at the indicated concentrations, and cells were collected at 60 min after the addition. Whole-cell extracts were prepared, and Rpb1 and PGK1 were detected by Western blot analysis as in panel A.

FIG. 5.

Rpb1 polyubiquitylation does not occur in UV- or 4-NQO-treated elc1Δ cells. (A) Rpb1 polyubiquitylation in UV-damaged wild-type and elc1Δ and rad7Δ mutant cells. Yeast strains harboring plasmid YEp112 that expresses HA-tagged wild-type ubiquitin were grown to an OD₆₀₀ of 1.0 in selective minimal medium, and ubiquitin expression from the CUP1 promoter was induced with 100 μM CuSO₄ for 2 h. After induction, cells in suspension were irradiated with UV (400 J/m²). Ubiquitylated proteins were purified with anti-HA resin (Sigma) and separated by SDS-6% PAGE, and Rpb1 ubiquitylation was analyzed with Rpb1-specific H14 antibody (Covance). (B) Rpb1 polyubiquitylation in 4-NQO-treated wild-type and mutant yeast cells. Methods were the same as described for panel A, except that cells were treated with 6 μg/ml of 4-NQO for 30 min. IP, immunoprecipitation; WB, Western blot; 0, untreated cells; +, cells treated with UV or 4-NQO.