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.

Nucleotide excision repair (NER) in eukaryotes is a versatile DNA repair process that functions in the removal of a large variety of DNA lesions, such as those induced by UV light or resulting from the addition of bulky chemical adducts to DNA bases (27, 31). In prokaryotes as well as eukaryotes, the repair of the DNA strand undergoing active transcription proceeds at a faster rate than the repair of the nontranscribed DNA strand (23, 24, 33). In humans, the preferential repair of the transcribed strand depends upon the CSA and CSB genes (34, 35), and in the yeast Saccharomyces cerevisiae, in addition to the requirement of RAD26 (37), which is the CSB counterpart, the Rpb9 subunit of RNA polymerase II (Pol II) (21) has also been shown to contribute to this repair pathway. Rpb9, a nonessential subunit of Pol II, is more effective in modulating the proficiency of repair in the coding region than in the region upstream of the transcription initiation site, whereas Rad26 contributes to the repair of both of these regions (21). The mechanism by which different protein factors contribute to the preferential repair of the transcribed strand in yeast or in humans is not understood.

Another phenomenon that occurs in response to DNA damage in both yeast and human cells is the polyubiquitylation of the largest Pol II subunit, Rpb1, which then leads to Pol II degradation by the proteasome (4, 7, 29). Although a role for the CSA and CSB proteins in Pol II polyubiquitylation and degradation was suggested from the observation that Pol II ubiquitylation and degradation do not occur in UV-damaged CSA or CSB fibroblasts (7), studies with yeast have failed to show a similar defect in UV-irradiated rad26Δ cells (39).

Genetic studies with yeast have indicated a requirement of the RAD7 and RAD16 genes for the repair of nontranscribed regions of the genome as well as for the repair of the nontranscribed DNA strand (36). The Rad7 and Rad16 proteins form a heterodimeric complex, named nucleotide excision repair factor 4 (NEF4) (13). Rad16 is a member of the SWI/SNF family of ATPases, and it also has a C₃HC₄ motif, a characteristic feature shared by E3 ubiquitin ligases. In addition, Rad16 possesses a C₄ motif, present also in proteins such as UvrA and Rad14, which show a preference for binding UV-damaged DNA (27, 31). The purified Rad7-Rad16 complex exhibits a DNA-dependent ATPase activity, but the presence of UV lesions in the DNA is inhibitory to this activity (12). Additionally, the Rad7-Rad16 complex displays a preference for binding UV-damaged DNA in an ATP-dependent manner (12, 13). We have previously suggested a role for the Rad7-Rad16 complex in the scanning of DNA for UV lesions, wherein using the energy of ATP hydrolysis, the enzyme translocates along the DNA, but at the site of a DNA lesion, the enzyme stops its translocation because of the inhibition of its ATPase activity (12). The lesion-bound Rad7-Rad16 complex then would serve as the nucleation site for the recruitment of other NER factors.

Yeast Elc1 is a homolog of mammalian elongin C (2), which forms a heterotrimeric complex with elongins A and B (3). The mammalian elongin A, B, C complex increases the rate of transcription elongation by suppressing Pol II pausing (5, 6). In addition to the involvement of this complex in transcription elongation, mammalian elongins B and C exist in a complex with cullin 2, the RING-H2 finger protein Rbx1, and the VHL (Von Hippel-Lindau) protein (8, 16, 18, 19, 22, 26). This protein complex, named VHL complex, bears a strong similarity to SCF (Skp1-Cullin-F-box) ubiquitin ligase, as elongin C shares sequence homology with Skp1, elongin B is a ubiquitin-like protein, and the VHL protein functions in a manner akin to F-box proteins, which bind the protein substrate targeted for ubiquitylation (8, 16-19, 22, 26).

A role for yeast Elc1 as a component of an E3 ubiquitin ligase has been suggested from the fact that Elc1 physically associates with the Rad7-Rad16 proteins (28). In this complex, Rad16 is a RING domain protein similar to Rbx1 in VHL, and Rad7 has a SOCS box motif, present in VHL and in other substrate-binding subunits of E3 enzymes. A role for the Elc1-Rad7-Rad16 complex in regulating the levels of Rad4 protein has been indicated from the observation that Rad4 levels rise in undamaged rad7Δ, rad16Δ, and elc1Δ yeast cells (28).

Here we provide evidence for a novel role of Elc1 in Pol II polyubiquitylation and its degradation in response to DNA damage in yeast cells. Unexpectedly, however, the Rad7 and Rad16 proteins have no role in modulating this Pol II modification. Our observations raise the strong possibility that, in addition to being a part of the Rad7-Rad16-Elc1 ubiquitin ligase, Elc1 is a component of a novel ubiquitin ligase which plays a key role in Pol II polyubiquitylation and degradation in DNA-damaged yeast cells. We discuss our findings in relation to previous observations that have been made in yeast cells for Pol II degradation.

MATERIALS AND METHODS

Yeast strains.

The strains used in this study are derivatives of the wild-type strain YRP668 (MATa his3-Δ1 leu2-3,−112 trp1Δ::URA3⁺ ura3-52) and are as follows: EMY74-7, MATa his3-Δ1 leu2-3,112 trp1Δ ura3-52; YRP669, elc1Δ; YR14-42, rad14Δ; YR14-167, rad14Δ elc1Δ; YR5-50, rad5Δ; YR 5-204, rad5Δ elc1Δ; YR18-53, rad18Δ; YR18-109, rad18Δ elc1Δ; YR52-88, rad52Δ; YR52-95, rad52Δ elc1Δ; YR7-50, rad7Δ; YR16-24, rad16Δ; YR7-73, rad7Δ elc1Δ; YR16-56, rad16Δ elc1Δ; YR26-1, rad26Δ; YR26-124, rad26Δ elc1Δ; YR23-23, rad23Δ; YR23-25, rad23Δ elc1Δ. Standard genetic techniques were used for construction and growth of strains.

Deletion of ELC1 gene.

A disruption cassette method (1) was used to delete the entire open reading frame of the ELC1 gene. Following transformation of the haploid strain EMY74-7 with the disruption cassette obtained after digestion of plasmid pPM1120 with EcoRI and Sph1, Ura3⁺ progeny colonies were screened for the presence of the elc1 deletion by examining the integration pattern by PCR with primer pairs spanning the presumptive recombination site.

UV sensitivity assays.

Cells grown in yeast-peptone-dextrose (YPD) were harvested in exponential phase, centrifuged, and suspended in water at a density of 2 × 10⁸ cells/ml. Sequential 10-fold serial dilutions were made, and 5 μl of each dilution was spotted onto YPD plates. When the spots had dried, the plates were UV irradiated, followed by incubation at 30°C in the dark.

UV and 4-nitroquinoline-1-oxide (4-NQO) treatment of yeast cells.

For UV treatment, an overnight YPD culture was inoculated into YPD medium to give an optical density at 600 nm (OD₆₀₀) of 0.2. When the culture had reached an OD₆₀₀ of 1.0, cells were centrifuged and resuspended in an equal volume of phosphate-buffered saline, keeping the OD₆₀₀ at 1.0. To UV irradiate cells, a 50-ml suspension of cells was placed in a large petri dish (150 by 15 mm), stirred vigorously and continuously with a magnetic mixer, and exposed to UV light (400 J/m²). Cells were suspended in 50 ml of 2× YPD medium in a flask and placed in the dark at 30°C in a shaking water bath. Aliquots of cells were removed at the indicated times for protein extraction.

For NQO treatment, yeast cells in logarithmic phase (as described above) grown in YPD were treated with 4-NQO by adding appropriate volumes of a 10-mg/ml stock solution of 4-NQO dissolved in dimethyl sulfoxide to the culture medium, followed by incubation for 30 min. After treatment, cells were centrifuged, washed, and processed for protein extraction.

Preparation of yeast cell extracts.

Yeast cells treated with UV or 4-NQO were harvested and resuspended in Y-PER lysis buffer (Pierce, Rockford, IL) to which a protease inhibitor mixture (Mini-Complete; Roche, Mannheim, Germany), phosphatase inhibitor cocktail II (Sigma), and 10 mM N-ethylmaleimide (Sigma) were added at 1 ml Y-PER buffer/g cell pellet. The cells were broken with glass beads using a Mini-Bead Beater (Biospec, Bartlesville, Oklahoma) to yield greater than 90% broken cells. Cellular debris and unbroken cells were removed by centrifugation at 20,000 × g for 10 min. The protein concentration was determined by the Bio-Rad protein assay. Equivalent protein amounts (25 μg) were loaded on a 4 to 20% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel (precast gel; Bio-Rad). Western analysis for Rpb1 was done using monoclonal antibody (MAb) 8WG16 (Promega) mouse RNA polymerase II antibody. As a loading control, PGK1 levels (phosphoglycerate kinase 1) were examined in the same gel using mouse MAb PGK1 (Molecular Probes).

In vivo ubiquitylation of Rpb1.

Yeast strains were transformed with either plasmid YEp112, encoding an epitope from hemagglutinin (HA) of influenza virus attached to the amino terminus of ubiquitin, or with YEp113, which is same as YEp112 except that it contains a mutation of Lys-48 to Arg-48 in ubiquitin. In these plasmids, the HA-ubiquitin coding sequence is under the control of the copper-inducible CUP1 promoter (14). Transformed cells were grown from an OD₆₀₀ of 0.2 to an OD₆₀₀ of 1.0 in selective minimal medium, induced with 100 μM CuSO₄ for 2 h, and than treated with 4-NQO (6 μg/ml) for 30 min. For UV treatment, 50 ml of cells was placed in a 150- by 15-mm petri dish and irradiated, and yeast cell extracts were prepared as described above. The cleared cell extracts were incubated for 2 h with prewashed anti-HA affinity gel (EZview Red anti-HA affinity gel; Sigma) and washed two times with the same Y-PER lysis buffer as used for the preparation of cell extracts (see above). The affinity gel was then resuspended in the same volume of 2× SDS loading buffer and boiled for 5 min. The eluted proteins were loaded onto an SDS-6% polyacrylamide gel and electrophoresed for 3 h at 150 V. Ubiquitylated Pol II was visualized by Western analysis using mouse RNA polymerase II and H14 monoclonal antibody (Covance).

RESULTS

Epistasis of ELC1 with genes in the NER epistasis group.

Because of the physical association of Elc1 with the Rad7-Rad16 protein complex (28), we first examined whether the elc1Δ mutation exhibits an epistatic relationship with genes of the NER epistasis group. For this purpose, we combined the elc1Δ mutation with the rad14Δ mutation defective in NER, the rad18Δ or rad5Δ mutation defective in replication promotion through DNA lesions, and the rad52Δ mutation defective in recombination and examined the UV sensitivity of the corresponding single and double mutants. As shown in Fig. 1, while the UV sensitivity of rad14Δ elc1Δ was the same as that of rad14Δ, the UV sensitivity of the rad18Δ, rad5Δ, or rad52Δ mutations is enhanced when they are combined with elc1Δ. These observations suggested the possible involvement of Elc1 in NER.

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.

Next, we examined the effect of the elc1Δ mutation on the UV sensitivity of the rad7Δ and rad16Δ mutations, which confer defects in the repair of nontranscribed regions of the genome as well as in the repair of the nontranscribed DNA strand, and on the UV sensitivity of the rad26Δ mutation, defective in the preferential repair of the transcribed strand. Whereas the UV sensitivity of rad7Δ or rad16Δ was not affected upon the introduction of elc1Δ into either of these mutant strains (Fig. 2), the rad26Δ elc1Δ double mutant displayed a higher degree of UV sensitivity than the rad26Δ or elc1Δ single mutant (Fig. 3). These observations support a role for Elc1 in Rad7-Rad16-dependent NER.

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.

Epistasis of rad23Δ mutation with the elc1Δ mutation.

Rad23 functions in NER in several ways. It is a component of NEF2, comprised of the Rad4-Rad23 heterodimer (10), which shows an affinity for binding UV-damaged DNA (11, 15). Also, Rad23 promotes complex formation between Rad14 and TFIIH (9). Furthermore, the ubiquitin-like domain present at the N terminus of Rad23 (38) promotes interactions with the proteasome (32), and it has been suggested that Rad23 interaction with the 19S proteasomal complex increases the rate of NER through a chaperone-like role of the ATPases present in the 19S complex (30).

To check for a possible involvement of Elc1 in some aspect of Rad23-dependent modulation of NER, we determined whether the UV sensitivity of the rad23Δ mutant was affected upon the introduction of the elc1Δ mutation. As shown in Fig. 2, since the UV sensitivity of the rad23Δ elc1Δ double mutant was the same as that of the rad23Δ mutation, an epistatic relationship between the Rad23 and Elc1 functions is indicated. In Discussion, we elaborate upon the significance of this observation for the possible involvement of the Rad7-Rad16-Elc1 complex in promoting the turnover of the NER ensemble in a Rad23-19S proteasomal complex-dependent reaction.

Requirement of ELC1 for RNA polymerase II degradation in response to DNA damage.

In looking for a role for Elc1 in promoting NER, we first considered the possibility that the Rad7-Rad16-Elc1 complex functions as an E3 ubiquitin ligase which regulates the levels of NER proteins. While these studies were in progress, a report appeared indicating a role for this protein complex in regulating the levels of Rad4 in undamaged yeast cells (28).

Quite unexpectedly, we have uncovered a role for Elc1 in promoting Pol II degradation in DNA-damaged yeast cells. As shown in Fig. 4A, Rpb1 levels decline in UV-treated wild-type cells, and we find that reduction in Rpb1 levels occurs much more rapidly and is much more extensive in rad14Δ cells than in wild-type cells. This is to be expected, however, since in the absence of any removal of UV lesions, Pol II would be subjected to stalling at the lesion sites to a much higher degree in rad14Δ cells than in wild-type cells, which would then lead to a faster and higher level of Pol II degradation in rad14Δ cells. In striking contrast to the wild-type or rad14Δ strain, we find that, in the elc1Δ strain, Rpb1 levels did not decline in UV-damaged cells. However, and surprisingly, Rpb1 levels declined in UV-damaged rad7Δ and rad16Δ cells, and in keeping with the intermediate level of UV sensitivity of these strains, the level of reduction in Rbp1 levels that occurred in these strains was intermediate between that seen in the wild-type strain and the rad14Δ strain.

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.

To ascertain that the effect of the elc1Δ mutation on Rpb1 degradation was not limited to UV damage, we examined the levels of Rpb1 in cells treated with 4-NQO. As shown in Fig. 4B, in contrast to wild-type cells, where a decline in the level of Rpb1 is observed after treatment with 4 or 6 μg/ml of NQO, Rpb1 levels remained unchanged in NQO-treated elc1Δ cells.

From these observations, we conclude a role for Elc1 in promoting Pol II degradation in DNA-damaged yeast cells; the Rad7 and Rad16 proteins, however, do not affect this process.

Elc1 is required for Rpb1 polyubiquitylation in DNA-damaged yeast cells.

Rpb1 ubiquitylation was examined in wild-type and mutant yeast strains carrying the plasmid YEp112, which expresses ubiquitin tagged with the HA epitope. Yeast strains were treated with UV or 4-NQO, and the ubiquitylated proteins were purified by binding the cellular proteins from lysed yeast cells to an anti-HA resin, followed by separation by SDS-polyacrylamide gel electrophoresis (PAGE). Ubiquitylated Rpb1 was detected by Western analyses using Rpb1-specific H14 antibodies. As shown in Fig. 5A, Rpb1 becomes polyubiquitylated in UV-treated wild-type cells and also in rad7Δ cells, but not in elc1Δ cells. Furthermore, whereas extensive Rpb1 polyubiquitylation was observed in NQO-treated wild-type cells as well as in rad14Δ, rad7Δ, and rad16Δ cells, no Rpb1 polyubiquitylation could be discerned in elc1Δ cells (Fig. 5B). From these observations, we infer a requirement of Elc1 for Rpb1 polyubiquitylation.

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.

Rpb1 polyubiquitylation occurs through lysine-48-linked ubiquitin chains.

Next, we verified that the Rpb1 polyubiquitylation observed in our experimental conditions, in fact, results from lysine-48-linked ubiquitin chains, which mark proteins for proteasomal degradation. To inactivate the formation of lysine-48-linked polyubiquitin chains, we introduced plasmid YEp113, which expresses the K48R mutant ubiquitin, into the wild-type, rad7Δ, and rad16Δ strains. As expected, no Rpb1 polyubiquitylation was observed in 4-NQO-treated wild-type cells or in rad7Δ or rad16Δ cells (data not shown). Hence, the Rpb1 polyubiquitylation we observe in wild-type cells or in rad7Δ or in rad16Δ cells is mediated through lysine-48-linked ubiquitin chains.

DISCUSSION

Genetic studies with the yeast ELC1 gene have revealed the involvement of this gene in at least two separate cellular processes. In one of these, we suggest Elc1 to be a structural and functional component of the Rad7-Rad16-dependent ubiquitin ligase and to modulate the efficiency of NER, and in the other, we propose Elc1 to be a component of a different ubiquitin ligase which promotes Pol II polyubiquitylation and its degradation in DNA-damaged yeast cells. Below, we summarize the evidence in support of these two Elc1-dependent ubiquitin ligases and discuss their respective roles in Rad7-Rad16-dependent NER and in Pol II ubiquitylation and degradation.

A role for Elc1 in NER is suggested by the fact that the UV sensitivity of the rad14Δ elc1Δ double mutant is similar to that of the totally incision-defective rad14Δ mutation. More importantly, the epistatic interaction of the elc1Δ mutation with the rad7Δ and rad16Δ mutations and the synergistic enhancement in UV sensitivity when the elc1Δ mutation is combined with rad26Δ are suggestive of a role for Elc1 in the Rad7-Rad16-dependent subpathway of NER which is specific for the repair of lesions from the nontranscribed regions of the genome. These genetic observations suggesting a role for Elc1 in combination with the Rad7 and Rad16 proteins are also consistent with the evidence of a physical association among these three proteins.

A previous study has indicated a role for the Rad7-Rad16-Elc1 protein complex in modulating the levels of Rad4 protein in undamaged yeast cells (28). However, since Rad4 is indispensable for NER (10), and since any cellular conditions that increase the stability of Rad4 lead to increased UV resistance (25), the promotion of Rad4 degradation by the Rad7-Rad16-Elc1 ubiquitin ligase fails to explain how such an action of this complex could have a positive effect upon NER. For that to happen, the Rad7-Rad16-Elc1 ligase would have to modulate NER in some other more positive manner.

The 26S proteasome, composed of a 19S regulatory complex and a 20S complex, promotes the degradation of polyubiquitylated proteins in an ATP hydrolysis-dependent manner. A role for the 19S complex, which is comprised of six homologous ATPases of the AAA family, has been indicated from the observation that inhibition of ATPase activities of the 19S complex inhibits NER in cell-free yeast extracts and that mutations in the ATPase subunits of the 19S complex confer increased UV sensitivity (30). In addition, genetic studies have indicated a role for the ubiquitin-like domain of Rad23 in the recruitment of the 19S complex to the NER ensemble at the damage sites (30). In experiments that have been carried out in our laboratory to examine the role of the 19S proteasomal complex in NER, we have not been able to uncover any evidence for the direct involvement of the 19S complex in NER, as we find that the addition of purified 19S complex to the NER system reconstituted from highly purified yeast proteins has no stimulatory effect on the proficiency of incision of UV-damaged DNA. In the absence of any evidence for a direct role of the 19S complex in NER, we now consider it more likely that some of the NER protein(s) have to become ubiquitylated before they can be acted upon by the 19S complex. In such a reaction, the Rad7-Rad16-Elc1 ubiquitin ligase could effect the ubiquitylation of NER proteins which are then acted upon by the ATPases present in the 19S complex and displaced from the lesion site. The efficient turnover of the NER ensemble from the lesion sites would lead to an increase in the proficiency of NER. But, for that to happen, we expect there to be no lysine-48-linked polyubiquitylation of NER protein(s), as the resulting protein degradation would lead to a decrease rather than an increase in the proficiency of NER.

Importantly, we provide evidence here for a role of Elc1 in promoting lysine-48-linked polyubiquitylation of Rpb1 and its subsequent degradation in DNA-damaged yeast cells. In this role, Elc1 functions independently of Rad7 and Rad16. We posit that Elc1 functions in this role as a component of another ubiquitin ligase which promotes Pol II polyubiquitylation and degradation. This ubiquitin ligase complex could be analogous to the human VHL complex, which is comprised of the cullin 2, elongins B and C, and Rbx1 proteins.

Our observation that Elc1 is indispensable for Pol II ubiquitylation and degradation and our suggestion that it functions in this role as a component of a VHL-like ubiquitin ligase would seem to be at odds with a previously published study that has implicated the requirement of Rsp5 ubiquitin ligase in Pol II ubiquitylation and degradation in DNA-damaged yeast cells (4). Since RSP5 is an essential gene, the studies linking Rsp5 to damage induced Pol II degradation had to be done in a temperature-sensitive (rsp5-1) mutant. In these studies, whereas Rpb1 polyubiquitylation and degradation were shown to be induced by 4-NQO in a wild-type yeast strain at 37°C, no polyubiquitylation or loss of Rpb1 was seen to occur in the rsp5-1 mutant at 37°C (4). However, recent observations indicating that Rsp5 function is important for maintaining proper ubiquitin levels in yeast cells (20) have raised the strong possibility that the lack of Pol II polyubiquitylation and degradation in the rsp5-1 mutant at 37°C results from the severe depletion of ubiquitin levels that occur in this mutant at the restrictive temperature and not because Rsp5 ubiquitin ligase function is needed for Pol II ubiquitylation.

In summary, here we identify a novel role for Elc1 in Pol II ubiquitylation and degradation in DNA-damaged yeast cells and suggest the involvement of an Elc1-dependent ubiquitin ligase in this reaction. In addition, we raise the possibility of a role for Elc1 in increasing the proficiency of NER in nontranscribed DNA, where, as a component of Rad7-Rad16-Elc1 ubiquitin ligase, it could mediate the turnover of lesion-bound NER ensemble in a Rad23-19S proteasomal complex-dependent reaction. As a part of two distinct ubiquitin ligases, Elc1 would then contribute to the more proficient repair of the nontranscribed DNA strand, and it would modulate the repair of the transcribed strand by effecting the removal of lesion-bound Pol II.