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J. Biol. Chem., Vol. 279, Issue 29, 29875-29878, July 16, 2004

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DNA Damage-induced Def1-RNA Polymerase II Interaction and Def1 Requirement for Polymerase Ubiquitylation in Vitro^*

James Reid and Jesper Q. Svejstrup

From the Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms, Hertfordshire EN6 3LD, United Kingdom

Received for publication, April 26, 2004 , and in revised form, May 14, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
UV-induced DNA damage results in ubiquitylation and degradation of RNA polymerase II (RNAPII). In yeast, this requires the DEF1 gene, the product of which forms a complex with the transcription-coupling repair factor, Rad26. However, whether Def1 is directly involved in RNAPII ubiquitylation has remained unclear. Here we report the establishment of a reconstituted system for studying UV-induced RNAPII ubiquitylation, which mimics the known requirements for this process in vitro. Using this system, we show that Def1 is indeed directly required for RNAPII ubiquitylation. Moreover, Def1 interacts with RNAPII in a damage-dependent manner. These results support a model in which Def1 interacts with RNAPII in response to DNA damage, recruiting the ubiquitylation machinery to enable its modification and subsequent degradation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
UV-induced DNA damage elicits a complex cellular response, which includes induction of genes required for repair, signaling to checkpoint proteins, and direct activation of various repair pathways (1). DNA lesions in the coding strand of active genes pose a particular threat to genome integrity and cellular survival, and multiple mechanisms have evolved to minimize the detrimental effects of such transcription obstacles (2). The best studied of these mechanisms is transcription-coupled DNA repair (TCR),¹ during which the transcribed strand of an active gene is repaired much faster than the nontranscribed strand and the genome in general (3, 4). The precise mechanism of TCR remains poorly understood but requires the protein products of genes that are mutated in patients suffering from the severe neurodegenerative disorder known as Cockayne's syndrome (5). The function in TCR of one of these proteins, Cockayne's syndrome B, is conserved from yeast to man (the yeast homologue is called Rad26) (6). Another transcription-relevant response to DNA damage is ubiquitylation and degradation of RNAPII (7–9). Ubiquitylation of RNAPII is mediated by the ubiquitin ligase Rsp5 (9, 10) and results in proteasome-dependent polymerase degradation (7, 9). Our recent work (11) uncovered an intriguing connection between TCR and RNAPII ubiquitylation in yeast; the TCR protein Rad26 forms a stable complex with a protein called Def1 when isolated from chromatin. Like yeast cells lacking RAD26, def1 cells are no more sensitive than wild type cells to UV irradiation, but DEF1 deletion dramatically exacerbates the damage sensitivity of cells compromised for nucleotide excision repair (i.e. rad16 def1 mutants are much more UV-sensitive than rad16 single mutants). However, in contrast to rad26 cells, def1 cells have normal rates of both TCR and general DNA repair. These data suggest that Def1 is part of a pathway that represents an alternative to DNA repair. Most significantly, cells lacking the DEF1 gene are unable to ubiquitylate and degrade RNAPII in response to DNA damage. Taken together, these data suggest that Rad26-mediated TCR and Def1-mediated RNAPII ubiquitylation/degradation represent interconnected but distinct ways of contending with DNA damage in active genes (11).

The appealing model that resulted from the identification and initial genetic characterization of DEF1 (11, 12) predicts that Def1 is directly involved in RNA polymerase II ubiquitylation. However, biochemical evidence for this idea has been lacking. Resolving this issue is particularly pressing because def1 cells also have transcription defects (11), which raises the possibility that the effect of DEF1 deletion on ubiquitylation could be indirect. For example, Def1 might be required for the transcription of genes encoding ubiquitylation factors. Here we report the establishment of a reconstituted yeast in vitro system to study damage-induced protein ubiquitylation; we used this assay to show that Def1 protein is indeed directly involved in ubiquitylation of RNAPII. We also demonstrate the existence of a damage-dependent interaction between Def1 and RNAPII.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Plasmids and Strains—All Saccharomyces cerevisiae strains used in this study were grown and manipulated using standard techniques (16). The def1 and rad26 strains have been described previously (11). The Rsp5 temperature-sensitive strain, FW1808, and its parental strain, FY56, were kindly provided by Prof. Fred Winston (Harvard Medical School). Plasmids from which PCR products were derived for multiple HA tagging of Def1 and Rpb1 by homologous recombination were kindly provided by Kim Nasmyth (17). The strains used were JSY568 and JSY569 (W303 DEF1::URA3 MATa and MAT, respectively) (11); FY56 (MAT his4–192R5 lys2–128 ura3–52) and FW1808 (MAT his4–192R5 lys2–128 ura3–52 rsp5–1) (18); and JSY903 (W303 MATa DEF1–6HA::HIS3) and JSY919 (PY26 MATa trp1–1 RPB1–3HA::URA3) (this study).

A bacterial expression plasmid for Myc-tagged ubiquitin was constructed by amplifying a single ubiquitin open reading frame from the yeast genome with the Myc tag sequence incorporated into the 5' primer. The resulting PCR product was subcloned into the NdeI/BamHI sites of the expression vector pET-21c, and its genetic integrity was confirmed by sequencing.

Protein Purification—Recombinant Myc-tagged ubiquitin was purified using a procedure modified from (19). Briefly, the protein was expressed in Escherichia coli strain BL21(DE3). After induction, cells were resuspended in 20 mM Tris, pH 7.5, and 150 mM NaCl containing protease inhibitors (20) and lysed by sonication. The lysate was adjusted to pH 4.5 with acetic acid, and precipitated proteins were removed by centrifugation in a Sorvall SS-34 rotor at 10,000 rpm for 10 min at 4 °C. The supernatant was collected and adjusted to pH 5.1 with NaOH before applying the clarified extract to a Sepharose SP Fast Flow cation exchange column (Amersham Biosciences) equilibrated in 50 mM ammonium acetate, pH 5.1. The column was washed with 5 column volumes of the equilibration buffer before eluting the bound proteins in a linear gradient to 0.5 M ammonium acetate, pH 5.1. Myc-ubiquitin eluted at 300 mM ammonium acetate. Fractions containing ubiquitin were pooled and dialyzed against 20 mM Tris, pH 7.5, 50 mM NaCl and stored at –80 °C.

Def1-6HA and Rpb1-3HA were both purified using the same one-step affinity purification procedure. Yeast strains containing the tagged proteins were grown in 10-liter YPD cultures to a density of 2 x 10⁷ cells/ml. The cells were washed in 500 ml of ice-cold sterile water, and the cell pellet was resuspended in an equal volume of extraction buffer (0.2 M Tris, pH 7.5, 0.39 M ammonium sulfate, 10 mM MgSO₄, 20% glycerol, 1 mM EDTA, and 1 mM dithiothreitol plus protease inhibitors). Cells were disrupted under liquid nitrogen using a SPEX Certiprep freezer mill (Glen Creston), allowed to thaw on ice, and centrifuged at 35,000 rpm in a Beckman Ti45 rotor for 2 h at 4 °C. The supernatant was transferred to a fresh tube and recentrifuged before being applied to 0.5 ml of protein G beads coupled to the anti-HA antibody 12CA5. The cell lysate was incubated with the antibody beads at 4 °C with mixing for 4 h to overnight. The beads were washed extensively with 20 mM Tris, pH 7.5, 250 mM NaCl, 10% glycerol, and protease inhibitors. The HA-tagged protein was released from the beads by the addition of 5 mg/ml HA peptide (21) into the above buffer and incubation at 30 °C for 15 min. Eluates were snap-frozen in liquid nitrogen and stored at –80 °C.

In Vitro Ubiquitylation—Yeast strains were grown in cultures of up to 10 liters at 30 °C until they reached a density of 2 x 10⁷ cells/ml. For experiments with the temperature-sensitive mutant FW1808 (as well as its parent strain FY56), the cultures were then incubated for 1 further hour at the nonpermissive temperature (37 °C) (or at the permissive temperature as control) before harvesting and preparation of extracts. For a comparison between def1, rad26, and wild type extracts, the cells were resuspended in saline, UV-irradiated (100 J/m²), and allowed to recover in fresh YPD medium for 1 h at 30 °C before preparing extracts. Whole cell extracts were prepared as previously described (13).

For each 100 µl of in vitro ubiquitylation reaction, 200 µg of extract was incubated with 2 µg of Myc-ubiquitin, 1 µg of HA-tagged RNAPII, 1mM ATP, and 10 µM lactacystin in Ub reaction buffer (20 mM HEPES, pH 7.9, 10% glycerol, 60 mM KCl, 7 mM MgCl₂, plus protease inhibitors). The reactions were incubated at 30 °C for 1 h before being placed on ice and diluted with 200 µl of Nonidet P-40 buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, plus protease inhibitors). 10 µl of protein G beads coupled with the anti-Myc antibody 9E11 were then added to each reaction, and the mixture was incubated at 4 °C with mixing for 4 h to overnight. The beads were washed twice with 400 µl of Nonidet P-40 buffer, twice with 400 µl of Nonidet P-40 buffer with 1 M NaCl, and finally with Nonidet P-40 buffer again. Bound proteins were eluted by the addition of SDS-sample buffer and boiling for 5 min. The samples were loaded onto a 6% SDS-polyacrylamide gel, blotted, and probed with the anti-RNAPII C-terminal domain antibody 4H8, which recognizes the Ser-5 phosphorylated form of Rpb1, and separately with the anti-Myc antibody 9E10 to uncover total protein ubiquitylation. In the ubiquitylation assay using extracts from rsp5-ts (Fig. 2), the initial immunoprecipitation was performed with 4H8 antibody to immunoprecipitate RNAPII and the blot was then probed with 9E10 antibody to reveal ubiquitylation.

View larger version (25K): [in this window] [in a new window]   FIG. 2. In vitro ubiquitylation requires Rsp5. RNAPII ubiquitylation with extracts prepared from wild type (WT) or rsp5 strain. Experiments performed at the nonpermissive temperature were with extracts from cells that had been shifted to 37 °C for 1 h prior to preparation. In this experiment, RNAPII was immunoprecipitated with 4H8 antibodies, and polymerase ubiquitylation thereafter was detected with 9E10 antibodies (detection of Myc-tagged ubiquitin conjugates). This likely gave rise to the stronger reactivity of long ubiquitin chains (which have several epitopes per RNAPII molecule) in this assay compared with others (Figs. 1 and 3), where ubiquitylated proteins were first isolated by the use of anti-Myc antibodies followed by detection of ubiquitylated RNAPII by 4H8 antibody reactivity.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Reconstitution of RNAPII Ubiquitylation in Vitro—Our previous studies showed that nucleotide excision repair and RNAPII transcription can be faithfully reconstituted in vitro using crude cell-free yeast extracts. During preparation, these extracts are effectively depleted for nucleic acids so that no transcription or translation can take place (13). For the preparation of extract to reconstitute RNAPII ubiquitylation in vitro, cells were grown to mid-logarithmic phase and UV-irradiated prior to cell disruption (see "Materials and Methods" for details). Using such extracts in combination with recombinant Myc-tagged ubiquitin (endogenous ubiquitin is removed in the course of extract preparation) and highly purified HA-tagged RNAPII enabled us to establish an in vitro system for the study of RNAPII ubiquitylation (Fig. 1). RNAPII ubiquitylation in this reconstituted system required the addition of ATP and ubiquitin (Fig. 1B), and the ubiquitin chains conjugated to RNAPII in the extracts grew longer with increased incubation time (Fig. 1C) as expected. Even though UV irradiation of cells prior to extract preparations was routinely employed, this did not appear to be strictly required for the ubiquitylation activity of the extracts. Most of the experiments described under "Materials and Methods" were done with an incubation time during which only one or a few ubiquitin molecules could be conjugated to RNAPII. We conclude that ubiquitylation of RNAPII can be reconstituted in a cell-free system in vitro.

View larger version (53K): [in this window] [in a new window]   FIG. 1. An in vitro system to study RNAPII ubiquitylation in yeast. A, summary of the experimental procedure. IP, immunoprecipitation; ab, antibody. B, RNAPII ubiquitylation in vitro requires ATP and ubiquitin. Ubiquitylation reactions, performed as outlined in A, were fractionated by SDS-PAGE and probed with 4H8 antibodies to detect RNAPII. Asterisk, ubiquitylated RNAPII. C, time course of the reaction. Monoubiquitylated (asterisk) and polyubiquitylated RNAPII (RNAPII-ubi) are indicated.

Ubiquitylation in Vitro Requires Def1—Having established that the ubiquitylation system can indeed be used to reconstitute aspects of RNAPII ubiquitylation in vitro, we next investigated whether Def1 protein is directly required for this activity (Fig. 3). Previous data obtained with live cells had shown that DEF1, but not RAD26, is required for damage-induced RNAPII ubiquitylation and degradation, but whether the involvement of Def1 was direct was not assessed (11). Extracts from wild type, def1, and rad26 strains were prepared, and RNAPII ubiquitylation was compared (Fig. 3A). Extracts from wild type and rad26 cells supported robust ubiquitylation of RNAPII, whereas little, if any, RNAPII ubiquitylation was observed in def1 extracts (Fig. 3A, upper panel; compare lanes 3, 4, and 5). Importantly, the failure to ubiquitylate RNAPII in def1 extracts was not due to a general deficiency in protein ubiquitylation in the extract, as other proteins were modified as efficiently in the def1 extract as in wild type and rad26 extracts (Fig. 3A, lower panel). This indicates that Def1 is specifically required for ubiquitylation of RNAPII.

View larger version (33K): [in this window] [in a new window]   FIG. 3. Ubiquitylation in vitro requires Def1. A, RNAPII ubiquitylation with extracts prepared from wild type (WT), def1, and rad26 strains. Upper panel, membrane probed with 12CA5 antibodies (RNAPII detection); lower panel, membrane probed with 9E10 antibodies (detection of total ubiquitylated (ubi) protein). B, purified Def1–6HA protein. C, addition of purified Def1 protein to def1 extracts recovers RNAPII ubiquitylation activity. Upper panel, membrane probed with 12CA5 antibodies (RNAPII detection); lower panel, membrane probed with 9E10 antibodies (detection of total ubiquitylated protein). M, molecular weight markers.

Def1 Interacts with RNAPII in a Damage-dependent Manner—The effect of Def1 on RNAPII ubiquitylation might conceivably be achieved through direct interactions with the polymerase. Such interactions might be constitutive, but they could also be induced by DNA damage. To examine these possibilities, cells expressing an epitope-tagged version of Def1 were grown to mid-logarithmic phase and UV-irradiated. Extracts were prepared at different times during recovery, and Def1 was immunoprecipitated. Finally, the presence of RNAPII in the precipitates was examined by Western blotting (Fig. 4). A low but clearly detectable amount of RNAPII co-immunoprecipitated with Def1 in the absence of UV irradiation (Fig. 4B, lane 1). However, a dramatic increase in the amount of RNAPII associated with Def1 was observed shortly after UV irradiation (Fig. 4B, lanes 3 and 4). As cells recovered after UV irradiation, detectable interaction between Def1 and RNAPII also decreased (Fig. 4B, compare lanes 4, 5, and 6). No immunoprecipitation signals were obtained from side-by-side control experiments with cells expressing untagged Def1 (data not shown). Only a small proportion of the total RNAPII within these extracts was precipitated by Def1 under these conditions (data not shown).

View larger version (30K): [in this window] [in a new window]   FIG. 4. Def1 interacts with Def1 in a damage-dependent manner. A, outline of experimental approach. IP, immunoprecipitation. B, immunoprecipitation of Def1 before and after UV irradiation of DEF1–6xHA cells. Results from Western blotting using 4H8 antibodies to detect co-precipitated RNAPII (RNAPII) and 12CA5 antibodies to detect precipitated HA-tagged Def1 (Def1-HA) are shown.

The data shown here are notable for several reasons. First, they demonstrate that Def1 protein is indeed directly involved in RNAPII ubiquitylation. Our findings thus support the idea that Def1 interacts with RNAPII in response to DNA damage, recruiting the ubiquitylation machinery to enable its modification and subsequent degradation. The exact manner in which this occurs remains unknown, but preliminary experiments with purified Def1 and Rsp5 suggest that these proteins do not interact directly.² In general, questions about the mechanism of action of Def1 await the development of a purified RNAPII ubiquitylation system, which is presently in progress. Second, the cell-free ubiquitylation assay used here is likely to be generally applicable for studying UV-induced protein modification. A mammalian cell-free system for studying RNAPII ubiquitylation has also been reported (14, 15). Because the yeast extracts we employ also support DNA repair and transcription under similar conditions (13), the procedure potentially can be used to more generally study damage responses. This is particularly attractive because of the extraordinary genetic tractability of S. cerevisiae, in which numerous mutants with defects in various repair and checkpoint pathways have been isolated.

	FOOTNOTES

 
^* This work was supported by a grant from Cancer Research UK (to J. Q. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Fax: 44-207-269-3801; E-mail: j.svejstrup{at}cancer.org.uk.

¹ The abbreviations used are: TCR, transcription-coupled DNA repair; RNAPII, RNA polymerase II; HA, hemagglutinin; YPD, yeast extract-peptone-dextrose.

² J. Reid and J. Q. Svejstrup, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Prof. Fred Winston for supplying the rsp5 temperature-sensitive yeast strain.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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This Article Abstract Full Text (PDF) All Versions of this Article: 279/29/29875    most recent C400185200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Reid, J. Articles by Svejstrup, J. Q. Search for Related Content PubMed PubMed Citation Articles by Reid, J. Articles by Svejstrup, J. Q. Social Bookmarking                      What's this?