Ultraviolet radiation-induced ubiquitination and proteasomal degradation of the large subunit of RNA polymerase II. Implications for transcription-coupled DNA repair.

We have shown previously that UV radiation and other DNA-damaging agents induce the ubiquitination of a portion of the RNA polymerase II large subunit (Pol II LS). In the present study UV irradiation of repair-competent fibroblasts induced a transient reduction of the Pol II LS level; new protein synthesis restored Pol II LS to the base-line level within 16-24 h. In repair-deficient xeroderma pigmentosum cells, UV radiation-induced ubiquitination of Pol II LS was followed by a sustained reduction of Pol II LS level. In both normal and xeroderma pigmentosum cells, the ubiquitinated Pol II LS had a hyperphosphorylated COOH-terminal domain (CTD), which is characteristic of elongating Pol II. The portion of Pol II LS whose steady-state level diminished most quickly had a relatively hypophosphorylated CTD. The ubiquitinated residues did not map to the CTD. Importantly, UV-induced reduction of Pol II LS level in repair-competent or -deficient cells was inhibited by the proteasome inhibitors lactacystin or MG132. These data demonstrate that UV-induced ubiquitination of Pol II LS is followed by its degradation in the proteasome. These results suggest, contrary to a current model of transcription-coupled DNA repair, that elongating Pol II complexes which arrest at intragenic DNA lesions may be aborted rather than resuming elongation after repair takes place.

dation in the proteasome, this modification enables cells to control the level of many regulatory proteins (1)(2)(3). Some ubiquitinated proteins, however, are not targeted to the proteasome (4 -6).
Ubiquitination plays a role in the response of the cell to DNA damage including the accumulation of p53 (7,8). Mammalian cells with a temperature-sensitive E1 show increased sensitivity to ultraviolet (UV) radiation when grown at the non-permissive temperature (9). The yeast RAD6 gene encodes an E2 (10), and the product of the yeast RAD23 gene possesses an NH 2 -terminal ubiquitin-like domain that is essential for its role in DNA repair (11). Lastly, a proteasomal subunit (SUG 1) copurifies with TFIIH, a multiprotein complex required for transcriptional initiation by RNA polymerase II (Pol II) as well as DNA repair via the nucleotide excision repair (NER) pathway (12,13).
7-10% of the large subunit of RNA polymerase II (Pol II LS) becomes ubiquitinated within minutes of exposing cultured human cells to UV radiation and the anti-cancer agent cisplatin (14). These two agents cause DNA lesions subject to repair by NER (7). Lesions induced by UV radiation and cisplatin can stop transcription by creating a physical block for the elongating Pol II apparatus when the lesions are located on the transcribed strand of a gene. Such lesions are repaired more rapidly by the NER pathway than are lesions located elsewhere in the genome. This preferential repair, called transcription-coupled repair (TCR), requires a functional Pol II LS (for review, see Ref. 13). Cells from patients with Cockayne syndrome (CS) demonstrate deficient TCR and hampered recovery of transcriptional activity after UV irradiation (15). Two complementation groups (CS-A and CS-B) have been identified, and in both cases the defective gene has been cloned (16,17). Interestingly, CS-A and CS-B cells also demonstrate deficient UVinduced ubiquitination of Pol II LS (14). A portion of Pol II binds to the CS-B protein (18).
During the transcription cycle, the COOH-terminal domain (CTD) of Pol II LS undergoes a cycle of phosphorylation/dephosphorylation (19,20). A relatively hypophosphorylated form of Pol II LS (IIa) preferentially binds to most promoters. Promoter clearance and transcript elongation are associated with phosphorylation of the CTD to yield a hyperphosphorylated (IIo) form. The CTD is an essential component of Pol II LS. It is comprised of a heptapeptide with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser repeated 52 times in mammalian cells. Many proteins involved in transcriptional regulation (21,22) as well as pre-mRNA processing (23)(24)(25) bind to the CTD, and it appears likely that the phosphorylation status of the CTD helps determine which proteins will bind (26). Studies of CTD phosphorylation have been assisted by the characteriza-tion of monoclonal antibodies (mAbs) that recognize distinct epitopes within the repeated heptapeptide (27,28). Some mAbs recognize a phosphorylated Ser residue (H5 or H14) and others recognize a dephosphorylated Ser residue (8WG16) within the repeated heptapeptide. 2 Because the status of Pol II LS after the cell has sustained DNA damage may affect NER as well as the recovery of transcription, we wished to determine the metabolic fate of Pol II LS subsequent to UV-induced ubiquitination. In the present study we establish that UV-induced ubiquitination of a portion of Pol II LS is followed by transient diminution of its steadystate level. Whereas the ubiquitinated Pol II LS molecules have a hyperphosphorylated CTD, the portion that appears to diminish most rapidly has a hypophosphorylated CTD. In repaircompetent fibroblasts, new protein synthesis permits recovery of base-line Pol II LS level. In repair-deficient xeroderma pigmentosum (XP) cells, the loss of Pol II LS is sustained. Proteasome inhibitors block the loss of Pol II LS in repair-competent or -deficient cells. The ubiquitinated residues are not located in the CTD.

EXPERIMENTAL PROCEDURES
Cell Culture-Cell culture was performed as described previously (14). The following XP fibroblast lines were used: DNA repair-competent, GM5659C and GM639D; XP-A, GM2009; XP-C, GM671; XP-D, GM8207; and XP-G, GM3021 from the National Institute of General Medical Sciences Human Genetic Mutant Cell Repository (Camden, NJ). They were maintained in Eagle's minimal essential medium (Sigma) supplemented with 15% fetal bovine serum, 2 mM L-glutamine, and 1 ϫ penicillin-streptomycin-neomycin (Sigma). UV irradiation at 254 nm was performed as described previously (14).
For individual experiments, the following agents were added to the cell culture media 1 h before UV irradiation and were maintained in the media after irradiation: cycloheximide, 20 mg/ml (Sigma); MG132, 1 M (Peptides International, Louisville, KY); and lactacystin, 5 M (Prof. E. J. Corey, Harvard).
The cell fractionation experiment was performed by extracting fibroblasts with TD buffer (0.5% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride) as described previously (27). Insoluble material was solubilized with SDS-PAGE sample buffer, and equicellular amounts of TD extractable and unextractable material were subjected to immunoblot analysis.
Mapping Pol II LS Ubiquitination Sites to a Non-CTD Domain-HeLa cells were stably transfected with two different constructs expressing ␣-amanitin-resistant Pol II LS: pHA-Wt, which expresses a hemagglutinin (HA)-tagged, ␣-amanitin resistant, full-length murine Pol II LS; and pHA-D31, which is identical to pHA-Wt except that the CTD has only 31 heptapeptide repeats such that all 8 CTD lysine residues are removed. The HeLa cells were transfected via lipofection with LipofectAMINE (Life Technologies, Inc.), and then cells expressing the ␣-amanitin-resistant Pol II LS were selected with 2 mg/ml ␣-amanitin. Clones of cells expressing pHA-Wt or pHA-D31 were then UV irradiated, allowed to recover at 37°C, and were subjected to lysis in hot 1% SDS, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride. Lysates were incubated in a boiling water bath for 5 min and then diluted with 6 volumes of cold 0.5% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride and placed in on ice. Immunoprecipitation was performed as described previously (14) with anti-HA mAb 12CA5 (Babco, Richmond, CA) non-covalently coupled to protein G-Sepharose beads (Pharmacia Biotech Inc.). Immunoprecipitated material was then subjected to immunoblot analysis with mAb H14.

UV Radiation Temporarily Reduces Pol II LS Level in Re-
pair-competent Cells-Because ubiquitinated proteins are often targeted to the proteasome, we investigated whether UVinduced ubiquitination of Pol II LS was followed by its degradation. Repair-competent fibroblasts were subjected to UV irradiation and allowed to recover at 37°C for 1-24 h. Whole cell extracts from equivalent numbers of cells were prepared and subjected to Western immunoblot analysis with H14, a mAb that binds specifically to Pol II LS (Fig. 1A, lanes  Repair-competent (GM639D) and XP-D (GM8207) fibroblasts were subjected to the indicated dose of UV radiation and then allowed to recover at 37°C for the indicated time intervals. Whole cell extracts were then subjected to 6% SDS-PAGE and immunoblot analysis with a panel of mAbs directed against distinct Pol II LS epitopes. Panel A, UV radiation induces ubiquitination of the hyperphosphorylated (IIo) form of Pol II LS and diminution of the hypophosphorylated forms (IIa). Immunoblots are with H14, a mAb that recognizes IIo, IIa, and Pol II LS forms with intermediate levels of CTD phosphorylation (lanes 1-12), or H5, a mAb specific for IIo (lanes [13][14][15][16][17][18][19][20][21][22][23][24]. Panel B, UV radiation induces a reduction in Pol II LS protein level rather than a change in CTD phosphorylation. Immunoblots are with mAb ARNA 3 (lanes 1-12) directed against a non-CTD epitope of Pol II LS or 8WG16 (lanes 13-24) directed against a non-phosphorylated CTD epitope. [1][2][3][4][5][6]. The H14 immunoreactive band from unirradiated cells is characteristically broad because H14 recognizes the hyperphosphorylated (IIo), hypophosphorylated (IIa), and intermediate forms of Pol II LS (Fig. 1A, lane 1). 1 h after a dose of UV radiation which would be expected to kill 10 -25% of repaircompetent fibroblasts (29,30), ubiquitinated forms of Pol II LS could be identified (Fig. 1A, lane 2). These forms migrate more slowly than IIo and have been demonstrated previously to represent ubiquitinated Pol II LS (14). In addition, the samples isolated 1-8 h after UV irradiation have a less broad Pol II LS band in which the relatively hypophosphorylated (IIa) forms appear preferentially diminished (lanes 2-4). By 16 -24 h after UV irradiation, the band width and intensity more closely resemble the unirradiated sample (lanes 5 and 6). This loss of Pol II LS was not appreciated in earlier studies (14) most likely because sublethally irradiated HeLa cells divide and resynthesize Pol II LS very rapidly. Similar results were obtained when cell extracts were normalized for total protein content (data not shown).
Because H14 recognizes a phosphorylated CTD epitope, 2 the UV-induced diminution of hypophosphorylated Pol II LS forms detected in Fig. 1A, lanes 2-4, could be caused by the altered steady-state level of the protein or altered phosphorylation status of the CTD. For example, if Pol II LS were acted upon by a kinase subsequent to UV irradiation, the broad Pol II LS band might be narrowed because of a shift of all Pol II LS forms toward IIo. To investigate why the Pol II LS band became narrower after UV radiation, the cell extracts were subjected to immunoblot analysis with different anti-Pol II LS mAbs. The level of hypophosphorylated (IIa) forms diminishes when the Pol II LS is detected with H14 as well as ARNA 3 (Fig. 1B, lanes  1-6), which recognizes a non-CTD epitope (31), or 8WG16 (Fig.  1B, lanes 13-18), which recognizes a non-phosphorylated CTD epitope (28). These data support a UV-induced loss of Pol II LS protein rather than a change in phosphorylation status.
In addition, the same samples were immunoblotted with H5, which is specific for the hyperphosphorylated (IIo) form of Pol II LS (27). The ubiquitinated forms reacted with H5 (Fig. 1A, lanes 14 -17) as well as with H14 (Fig. 1A, lanes 2-5), indicating that the ubiquitinated forms were also hyperphosphorylated even though the forms whose steady-state level diminishes after UV irradiation were relatively hypophosphorylated (see "Discussion").
UV Radiation Induces a Sustained Reduction of Pol II LS Level in XP-D Cells-We also performed a UV time course in an NER-defective xeroderma pigmentosum group D (XP-D) fibroblast cell line. In Fig. 1, A  Using pharmacologic agents that block the ability of the proteasome to degrade ubiquitinated proteins we demonstrated that the ubiquitinated Pol II LS was targeted to the proteasome. Both the specific and irreversible proteasomal inhibitor lactacystin (32) and the reversible albeit less specific inhibitor MG132 (33) led to less loss of Pol II LS 1-9 h after UV irradiation (Fig. 3, compare lanes 8 -11 with lanes 2-5 and  compare lanes 20 -23 with lanes 14 -17). In the absence of UV irradiation the inhibitors did not alter the level of Pol II LS (Fig. 3, lanes 6, 12, 18, and 24).
Characterization of the Ubiquitinated Fraction of Pol II LS-The ubiquitinated portion of Pol II LS was shown to be hyperphosphorylated (IIo) by virtue of its immunoreactivity with mAb H5 (Fig. 1A). Whereas many studies have shown that Pol II LS that is actively engaged in transcriptional elongation is in the IIo form, we have shown previously that a portion of IIo is also present in a transcriptionally quiescent extrachromosomal location where splicing factors are also located (23,27). These two different pools of IIo differ substantially in their ability to be extracted from mammalian nuclei with non-ionic detergents (27). The non-chromosomal (transcriptionally inactive) form is considerably more resistant to detergent extraction than the non-speckle-associated form. We demonstrated that the ubiquitinated form of Pol II LS is readily extractable with non-ionic detergent (Fig. 4). None of the ubiquitinated IIo was detected in the detergent-inextractable fraction, even when long exposures of the immunoblot were examined (data not shown). This is consistent with ubiquitinated IIo being the transcriptionally active (elongating) form of Pol II LS.
Mapping the Sites of Ubiquitination to a Non-CTD Domain-Because the ability of Pol II LS to be ubiquitinated appears to depend on the phosphorylation status of the CTD, we wished to determine whether ubiquitination was also localized to this important regulatory domain. The CTD of Pol II LS is comprised of 52 tandem repeats of a heptapeptide with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. All covalent modifications of Pol II LS besides ubiquitination, including serine/ threonine phosphorylation, tyrosine phosphorylation, and O-linked glycosylation, have been mapped to the CTD (34 -36). We therefore wished to determine whether ubiquitination maps to this domain of Pol II LS.
We were able to replace functionally the Pol II LS of HeLa cells with a truncated molecule in which all potential ubiquitination sites within the CTD were removed. Ubiquitination  1 h, lanes 7-12) and then UV irradiated and incubated at 37°C in the presence of cycloheximide (20 g/ml) for the indicated times. An identical time course was also performed without cycloheximide (lanes 1-5). Whole cell extracts were then subjected to 6% SDS-PAGE and immunoblot analysis with anti-Pol II LS mAb (H14). To control for the effect of cycloheximide, unirradiated samples were collected at the 24-h time point (ૺ, lanes 6 and 12).
occurs exclusively on lysine residues (1). The CTD includes 8 lysines all of which are located in the COOH-terminal third of the CTD (comprising heptapeptide repeats 34 -52) (37). Truncation of more than half of the CTD prevents its normal function, but truncation of the portion of the CTD including all 8 lysine residues yields a molecule capable of supporting a viable cell (38). Furthermore, a mutated variant of mouse Pol II LS has been cloned which will render cells resistant to the transcriptional inhibitor ␣-amanitin. This mutant bears a single amino acid change at a non-CTD amino acid (39). In a cell expressing both WT (human) Pol II LS and mutant (mouse) Pol II LS, only the mutant will function in the presence of ␣-amanitin (40). The amino acid sequences of WT mouse and human Pol II LS are nearly identical; there are only two sequence differences, neither of which involves CTD lysine residues (37).
We therefore isolated HeLa cells stably transfected with ␣-amanitin-resistant Pol II LS expression constructs bearing either full-length CTD with all 52 heptapeptide repeats (WT) or a truncated CTD with only 31 of 52 heptapeptide repeats which therefore lacked all 8 CTD lysine residues (⌬31). These constructs were HA epitope tagged so the product of the transfected construct could be separated from the host Pol II LS via specific immunoprecipitation. After UV irradiation, both WT and truncated HA-tagged Pol II LS molecules demonstrated ubiquitination thus proving that non-CTD lysine residues were being ubiquitinated (Fig. 5).

UV-induced Ubiquitination of Pol II LS in XP Cells-To address the question of whether UV-induced ubiquitination of
Pol II LS plays a mechanistic role in NER, we investigated Pol II LS ubiquitination in fibroblast cell lines established from patients with XP complementation groups A through G. Each complementation group lacks a functional gene product required for a mechanistic "step" of NER (7). In addition several forms yield defects in TCR (XP-B, -D, and -G) (13). If ubiquitination of Pol II LS plays a mechanistic role in NER or TCR, ubiquitinated Pol II LS might accumulate to unusually high levels in one or more of the XP subtypes, or the kinetics of the appearance/disappearance of ubiquitinated Pol II LS might be altered.
In Fig. 6A, UV time courses of two different repair-competent human fibroblast lines are shown (lanes 1-5 and 6 -10). Ubiquitinated forms of Pol II LS are induced within 1 h after UV irradiation and diminish thereafter until they become undetectable by 16 -24 h. In all XP cells, ubiquitination and deubiquitination occurred to an extent similar to that seen in repaircompetent cells and with similar kinetics (lanes 11-25). The  7-12). A control UV time course was carried out without added lactacystin (lanes 1-6). Panel B, XP-D fibroblasts were treated with the proteasomal inhibitor MG132 at 1 M for 1 h and then subjected to UV irradiation plus incubation at 37°C in the presence of MG132 for the indicated time intervals (lanes 19 -24). A control UV time course was carried out without added MG132 (lanes 13-18). To control for the affect of lactacystin and MG132, unirradiated samples were also collected at the 9 h time points (ૺ, lanes 6, 12, 18, and 24). In all cases, whole cell extracts were subjected to 6% SDS-PAGE and immunoblot analysis with anti-Pol II LS mAb (H14).

FIG. 4. The form of Pol II LS which is ubiquitinated is extractable with non-ionic detergents.
Repair-competent (639D) fibroblasts were subjected to the indicated dose of UV radiation and then allowed to recover at 37°C for the indicated time intervals. Cells were extracted with TD buffer containing 0.5% Triton X-100 and 0.5% sodium deoxycholate (see "Experimental Procedures"). The TD-unextracted material was solubilized in SDS-PAGE sample buffer, and equicellular amounts of soluble and insoluble material from each time point were subjected to 6% SDS-PAGE and immunoblot analysis with mAb H14.
FIG. 5. UV-induced ubiquitination does not occur on CTD lysine residues. HeLa cells were transfected with constructs expressing an HA epitope-tagged Pol II LS with a single amino acid change such that it is resistant to the transcriptional inhibitor ␣-amanitin (38). The constructs expressed either a Pol II LS with a full-length CTD (WT, 52 heptapeptide repeats) or a CTD truncated such that no lysine residues remained (⌬31-31 heptapeptide repeats). Stably transfected cell lines were selected by growth in ␣-amanitin as described under "Experimental Procedures." Panel A, two clones each of WT (6 and 8) and ⌬31 (1 and 2) were subjected to UV irradiation and allowed to recover at 37°C for 15 min. Plates of cells were then lysed, immunoprecipitated with anti-HA antibody 12CA5, and subjected to 6% SDS-PAGE and immunoblot analysis with mAb H14. Panel B, WT or ⌬31 cells were irradiated and allowed to recover for 1-24 h as indicated before being lysed, immunoprecipitated with anti-HA, and subjected to immunoblot analysis with mAb H14. The positions of IIo, IIa, and ubiquitinated forms of Pol II LS are indicated. The truncated IIo and IIa forms from ⌬31 migrated further into the SDS-PAGE gel than those of WT. results of XP-B, -D, -E, and -F cells (data not shown) were the same as those shown for XP-A and -G cells. These results do not support a mechanistic role for ubiquitination of Pol II LS in NER.
However, in all XP cell types in which no residual NER activity remains (i.e. all types except XP-C, see below) there is a sustained loss of Pol II LS forms less phosphorylated than IIo beginning by 1 h after UV irradiation and easily detectable by 8 h after UV irradiation (lanes 18 and 23). In fibroblasts with normal levels of NER and TCR there was a transient loss of less phosphorylated forms of Pol II LS at 1-8 h after UV irradiation (lanes 2, 3, and 7) which subsequently returned to base-line levels. Similarly in XP-C cells, which are deficient in "genome overall" NER but which maintain TCR (41), the level of less than maximally phosphorylated Pol II LS forms transiently diminishes but subsequently (and reproducibly) returns to at least base-line levels (lanes 12-15). Furthermore, when repaircompetent fibroblasts were subjected to a higher dose of UV radiation (which would be expected to kill most cells; see Ref. 30) the loss of less phosphorylated forms of Pol II LS was also sustained (lanes [27][28][29][30]. DISCUSSION This study establishes that UV radiation induces ubiquitination of a fraction of Pol II LS which is followed by the degradation of a fraction of Pol II LS in the proteasome. Another recent study identified a specific ubiquitin-protein ligase (Rsp5) capable of ubiquitinating Pol II LS in vitro and in vivo (43). The ability to inhibit the UV-induced reduction of Pol II LS steady-state level with MG132 or lactacystin, two proteasomal inhibitors, supports the conclusion that degradation takes place in the proteasome (Fig. 3).
Thus DNA repair-competent cells appear capable of temporarily down-regulating their Pol II LS level upon transcriptional arrest and then reaccumulating new Pol II LS when transcription resumes (Figs. 1 and 6). In repair-deficient cells or in those repair-competent cells subjected to a lethal (50 J/m 2 ) UV dose, Pol II LS levels remain low (Fig. 6). In XP-C cells, which maintain the ability to repair DNA damage and therefore recover transcriptional activity through TCR (they are only deficient in genome overall repair), Pol II LS levels also return to the base-line level within 16 -24 h (Fig. 6). Evidence for post-transcriptional regulation of the Pol II LS level already exists in Caenorhabditis elegans (44). In mammalian cells, ␣-amanitin and actinomycin D can also trigger Pol II LS degradation (45). Pol II LS is a component of the nuclear matrix, and its degradation may render DNA more susceptible to fragmentation of the sort observed during apoptosis (46).
The results of this study are complicated by the fact that multiple phosphorylated forms of Pol II LS exist. Although the form that gets ubiquitinated is highly phosphorylated, the forms whose steady-state level decreases most rapidly are less phosphorylated (e.g. see Fig. 1A). Because mAb H5, which is specific for highly phosphorylated Pol II LS (27), reacts with the ubiquitinated forms of Pol II LS (Fig. 1A, lanes 14 -17 and  20 -23) it can be concluded that the ubiquitinated form of Pol II LS is also highly phosphorylated. Furthermore, an antibody that recognizes a non-phosphorylated CTD epitope (mAb 8WG16) does not react with the ubiquitinated forms (Fig. 1B,  lanes 14 -17 and 20 -23). It should be noted that both H14 and 8WG16 recognize overlapping broad Pol II LS bands because the CTD of one Pol II LS molecule may have phosphorylated as well as non-phosphorylated heptapeptides. Presumably, the number and identity of phosphorylated versus non-phosphorylated residues among the approximately 250 serines, threonines, and tyrosines within the CTD determine the migration rate in SDS-PAGE.
A plausible interpretation of these steady-state results is that UV-induced ubiquitination and subsequent degradation of Pol IIo prevent its being recycled (via dephosphorylation) to the IIa form so that the steady-state level of IIa diminishes. The pool of IIa gets converted to IIo via the action of one or more CTD kinases. Some CTD kinases phosphorylate IIa to IIo during the normal transcription cycle, and others may be activated by heat shock or other cellular stresses (47,48). In the present study, some reduction in the level of Pol IIo did become apparent when resynthesis of Pol II LS was prevented with cycloheximide (Fig. 2) or when XP-D cells were irradiated so that transcription could not resume (see Fig. 1A).
Although the present study does not demonstrate directly that the fraction of Pol II LS which gets ubiquitinated and degraded is stalled at intragenic lesions, the results are consistent with this conclusion. It is known that UV-induced DNA lesions constitute a potent block to transcription at which elongating Pol II becomes stably arrested (49). The large subunit of arrested Pol II would be expected to exist in the IIo form. Western immunoblot analysis demonstrated that ubiquitinated Pol II LS reacts with mAb H5, which is specific for the IIo form. Although a previous study demonstrated a transcriptionally quiescent fraction of H5-immunoreactive Pol II LS present in structures called speckles or interchromatin granule clusters (27), we utilized the extractability of non-speckle-associated IIo with non-ionic detergent to strengthen the argument that the ubiquitinated IIo is not present in the speckles (Fig. 6). Furthermore, we have shown previously that UV-induced ubiquitination of Pol II LS is deficient in CS-A and CS-B fibroblasts, which have a defect in TCR. Thus it seems quite likely that elongating Pol II LS that arrests at intragenic DNA lesions becomes ubiquitinated and gets degraded in the proteasome. Future studies will be aimed at demonstrating directly that the stalled Pol II molecules get ubiquitinated.
The results presented here also establish that ubiquitination occurs on non-CTD lysine residues (Fig. 5). It will be of interest to determine whether the ubiquitinated lysine residues are located within DNA binding domains and whether ubiquitination per se promotes dissociation of the stalled Pol II from the DNA before Pol II LS degradation in the proteasome.
A current model of TCR in eukaryotes suggests that arrested transcripts back up, possibly assisted by elongation factor SII, then resume elongating after the lesion has been repaired (50, 51). This model is supported by the stability of DNA⅐Pol II⅐pre-mRNA ternary complexes arrested at intragenic lesions in in vitro DNA damage systems as well as by the belief that it would be metabolically wasteful for cells to abort partially elongated RNA transcripts. An important implication of the results presented in this study is that an elongating Pol II complex that arrests at an intragenic lesion will not finish elongating the interrupted transcript after repair takes place. If Pol II LS is ubiquitinated and degraded in the proteasome it suggests that the arrested transcript is aborted. Thus eukaryotic Pol II LS may behave as Escherichia coli RNA polymerase behaves. In E. coli the product of the mfd gene, TRCF, releases the arrested DNA⅐RNA polymerase⅐RNA ternary complex and recruits the bacterial NER apparatus (the UvrA 2 UvrB 1 complex) to the intragenic lesion (52).
Two lines of evidence (in addition to the results presented in this study) support the idea that SII, although essential for allowing Pol II to negotiate intrinsic pause sites, may be less important for TCR and/or the ability of cellular transcription levels to resume base-line levels after being arrested by UVinduced DNA damage. First, yeast with mutated SII does not demonstrate a deficiency in TCR (53), suggesting that SII is not essential for either the preferential repair of intragenic DNA lesions or the ability of transcription to resume once such lesions have been repaired. Second, a recent in vitro study leads to the conclusion that SII may be more effective at allowing Pol II to bypass lesions that cause the slowing of transcriptional elongation than allowing Pol II to bypass transcriptional arrest sites (54). In this study, a bulky N-2-acetylaminofluorene-DNA adduct located on the transcribed strand served as an absolute block to elongating Pol II. SII did not increase the ability of Pol II to transcribe through such a lesion. SII did however improve the ability of Pol II to read through a transcriptional pause site created by placing the N-2-acetylaminofluorene adduct on the non-transcribed DNA strand. In another study, N-2-acetylaminofluorene located on the transcribed strand promoted the dissociation of the stalled ternary Pol II⅐DNA⅐RNA complex (55). Furthermore, although an elegant in vitro study demonstrated that SII promoted the retrograde (3Ј to 5Ј) movement of Pol II from a T-T dimer, SII failed to displace stalled Pol II sufficiently to permit repair of the T-T dimer by photolyase (56).
The stalled Pol II complex appears to help recruit the repair apparatus to intragenic damage sites. It also appears to play a role in UV-induced signal transduction (57) and may inhibit basal transcription by binding up nuclear transcription factors such as the TATA box-binding protein, TBP (42). The ability to disassemble this complex, perhaps by degradation of Pol II LS, could be a critical step in the resolution of DNA damage response of the cell and the restoration of basal transcription after DNA repair is completed.