10.1128/MCB.01640-05.

2006, 26(11):4122. DOI:Mol. Cell. Biol. Ewa T. Lis and Floyd E. Romesberg 

DNA Damage ResponsecerevisiaeSaccharomycesRole of Doa1 in the

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MOLECULAR AND CELLULAR BIOLOGY, June 2006, p. 4122–4133 Vol. 26, No. 110270-7306/06/$08.00�0 doi:10.1128/MCB.01640-05Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Role of Doa1 in the Saccharomyces cerevisiae DNA Damage Response†Ewa T. Lis and Floyd E. Romesberg*

Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, California 92037

Received 23 August 2005/Returned for modification 24 October 2005/Accepted 16 March 2006

The cellular response to DNA damage requires not only direct repair of the damage but also changes in theDNA replication machinery, chromatin, and transcription that facilitate survival. Here, we describe Saccha-romyces cerevisiae Doa1, which helps to control the damage response by channeling ubiquitin from theproteosomal degradation pathway into pathways that mediate altered DNA replication and chromatin modi-fication. DOA1 interacts with genes involved in PCNA ubiquitination, including RAD6, RAD18, RAD5, UBC13,and MMS2, as well as genes involved in histone H2B ubiquitination or deubiquitination, including RAD6,BRE1, LGE1, CDC73, UBP8, UBP10, and HTB2. In the absence of DOA1, damage-induced ubiquitination ofPCNA does not occur. In addition, the level of ubiquitinated H2B is decreased under normal conditions andcompletely absent in the presence of DNA damage. In the case of PCNA, the defect associated with the doa1�mutant is alleviated by overexpression of ubiquitin, but in the case of H2B, it is not. The data suggest that Doa1is the major source of ubiquitin for the DNA damage response and that Doa1 also plays an additional essentialand more specific role in the monoubiquitination of histone H2B.

To maintain the genome, cells have evolved multiple path-ways to detect and respond to DNA damage. The cellularresponse to DNA damage has been particularly well charac-terized in the yeast Saccharomyces cerevisiae. An importantway in which this organism coordinates different facets of theDNA damage response is the posttranslational modificationof proteins. While phosphorylation has received a great deal ofattention, it has become increasingly clear that other types ofposttranslational modifications, such as ubiquitination, alsoplay critical roles. Protein ubiquitination is catalyzed by ubiq-uitin (Ub)-conjugating enzymes, which facilitate the formationof an isopeptide bond between the C terminus of Ub and alysine side chain of a substrate protein (41). Proteins may bemonoubiquitinated, or the Ub monomer may act as a point ofattachment for additional Ub monomers, resulting in poly-ubiquitination. The specific biological signal mediated by apolyubiquitin chain is determined, in part, by chain topology,which is differentiated by the Ub lysine residue used for chainextension (22, 32). K48-linked chains appear to target proteinsfor proteasomal degradation, whereas K63-linked chains ap-pear to regulate proteins involved in a wide range of processes,including DNA repair, mRNA translation, and endocytosis(10, 40). Ub modification of proteins is reversible; Ub may beremoved from proteins by deubiquitinating enzymes, whichhydrolyze the isopeptide bond between Ub and the substrateprotein, or by ubiquitin proteases, which remove Ub mono-mers from a polyubiquitin chain (2).

Under normal cellular conditions, Ub is produced as a fu-sion with ribosomal proteins; proteolytic cleavage from thesecarrier proteins generates a pool of free Ub (31), much ofwhich is used to form K48-linked Ub chains that target pro-teins for proteosomal degradation. Under conditions of stress,

it is thought that increased levels of Ub are supplied by theexpression of UBI4, which encodes a head-to-tail pentamer ofUb (9). However, no relationship between UBI4 and any facetof the DNA damage response has been established. While itssource remains to be more rigorously characterized, severalfunctions for Ub in the DNA damage response have beenidentified. For example, to effect changes in DNA synthesis,PCNA is monoubiquitinated by Rad6, with assistance fromRad18; it then may be K63 polyubiquitinated by Rad5 and theUbc13/Mms2 heterodimer (21, 40). These PCNA modifica-tions are thought to help control various pathways involved inthe repair of replication forks that have stalled or collapseddue to damaged DNA (21, 40). In association with Bre1 andLge1, Rad6 also monoubiquitinates H2B (25, 34, 45), whichplays a critical role in both the initiation and elongation ofRNA polymerase (Pol) II transcription (6, 19, 27, 48). WhileH2B ubiquitination is required under normal cellular condi-tions for transcription of certain genes, maintenance of H2Bubiquitination levels is also critical for the transcriptional andcell cycle response to DNA damage (15).

The doa1� mutant was originally identified in screens formutants that stabilize several normally short-lived proteins (14,20, 26). The observation that overexpression of Ub can com-plement the phenotypes led to the suggestion that Doa1 is aregulatory component of the proteasome. Doa1 contains fiveWD repeats, which are present in a number of eukaryoticproteins involved in cell cycle control, cell fate determination,transcription, transmembrane signaling, RNA metabolism, andvesicular trafficking (29). The N terminus of Doa1 is homolo-gous to PLAP (phospholipase A2-activating protein) (5) andLis-1 (a subunit of the platelet-activating factor acetylhydro-lase) (18), while the C terminus is not homologous to anyknown protein. High-throughput affinity precipitation studiesidentified a complex composed of Doa1, Cdc48 (AAA-ATPase),Rpa190 (RNA Pol I subunit), Rpa135 (RNA Pol I subunit),Rpo31 (RNA Pol III subunit), and Shp1 (regulator of Glc7 phos-phatase activity) (12, 14). Other studies showed that Doa1 binds

* Corresponding author. Mailing address: Department of Chemistry,10550 N. Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 784-7290.Fax: (858) 784-7472. E-mail: floyd@scripps.edu.

† Supplemental material for this article may be found at http://mcb.asm.org/.

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proteins containing UBX (Ub regulatory X) domains, such asUbx4, Ubx6, and Ubx7 (7, 43).

Doa1 was first implicated as being part of the DNA damageresponse in a genome-wide screen for genes involved in repair-ing UV- and MMS-induced DNA damage (17). More recently,it was shown that LUB1, the Schizosaccharomyces pombe ho-mologue of DOA1, plays a role in mediating the stress response(30). The temperature sensitivity of a lub1� mutant was ame-liorated by expression of Ub or the K63R Ub mutant but notby the expression of the K48R Ub mutant. The lub1� mutantalso exhibited decreased cellular levels of both Ub and poly-ubiquitin chains but no change in the mRNA levels of ubi4. Anincreased rate of Ub turnover was also observed. Based onthese observations, it was suggested that Lub1 is a negativeregulator of Ub degradation that acts to provide Ub specifi-cally for proteasome-mediated protein degradation.

In this report, we characterize the role of Doa1 in the S.cerevisiae DNA damage response. Interestingly, despite thefact that Doa1 and Lub1 are homologous proteins, they appearto play different roles in the stress responses of their respectiveorganisms. We present evidence that in S. cerevisiae, Doa1provides the majority of Ub used in the damage response. Wedemonstrate that DOA1 interacts with genes involved in bothPCNA and H2B ubiquitination and that in the absence ofDoa1, virtually no PCNA ubiquitination is induced in responseto DNA damage and levels of ubiquitinated H2B are notmaintained. Consistent with the localization of Doa1 to boththe cytoplasm and the nucleus (13, 24), the data suggest thatDoa1 is involved in the rapid recycling of Ub from theproteasomal degradation pathway into transcriptional anddamage response pathways.

MATERIALS AND METHODS

Media and general procedures. Yeast was cultured at 30°C in yeast extract-peptone-dextrose (YPD), synthetic complete, or Hartwell’s complete (HC) me-dium as described previously (39). Methyl methane sulfonate (MMS; Aldrich),hydroxyurea (HU; U.S. Biological), X-Gal (5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside; Amersham), 6-azauracil (6-AU; Fisher), Complete mini pro-tease inhibitor cocktail (Roche), antiubiquitin antibody (Sigma), anti-Flag anti-body (M2 mouse monoclonal; Sigma), anti-H3 trimethyllysine 4 antibody(ab8580; Abcam), antihemagglutinin (anti-HA) antibody (12CA5; Roche), an-tiphosphoglycerokinase (anti-PGK) antibody (22C5; Molecular Probes), anti-myc antibody (9E10; Santa Cruz), anti-mouse antibody–horseradish peroxidase(HRP) conjugate (Upstate), and anti-rabbit antibody–HRP conjugate (Upstate)were also used. Sequences of primers used in this study are available uponrequest.

Yeast two-hybrid screening. The LexA-based yeast two-hybrid system(DupLEX-A; Origene Technologies, Inc., Rockville, MD) was employed. DOA1was PCR amplified from S288C genomic DNA. The 5� primer contained an NcoIrestriction site and an polylinker encoding Gly-Gly-Ser-Gly-Gly. The 3� primercontained an XhoI restriction site. The PCR product was digested and ligatedinto NcoI/XhoI-digested pEG202 to generate a Doa1-LexA fusion. The Doa1-LexA-encoding plasmid, the reporter plasmid pSH18-34, and a genomic libraryin pJG4-5 (obtained from R. Brent, Molecular Sciences Institute, Berkeley, CA)were transformed into yeast EGY48; 1.5 � 106 transformants were screened.Activation of the LEU2 and lacZ reporter genes (located in EGY48 and pSH18-34, respectively) was used to identify physically interacting clones. Library plas-mids were isolated and sequenced to reveal the identities of the interactors. Thespecificity of the interactors was determined using pLexA-Max according to themanufacturer’s instructions. For direct yeast two-hybrid studies of Doa1-LexAwith Ub chains, DNA encoding one to five ubiquitins was amplified from S288Cgenomic DNA, using primers specific for UBI4. Isolated fragments were clonedinto the prey vector containing the B42 DNA activation domain. Interactionswere determined using the lacZ reporter as described above.

Strain construction and sensitivity to DNA damage. Yeast strains used in thisstudy are listed in Table 1. Double deletion mutants were constructed by trans-formation of the kanMX4 null deletion strains (Open Biosystems) with a double-stranded DNA deletion cassette specific for DOA1. Cassettes were generated byPCR amplification of the Kluyveromyces lactis LEU2 cassette from pUG73 or thekanMX6 cassette from pUG6, using gene-specific primers (16). Gene disruptionswere confirmed by PCR, as well as by phenotypic screening of multiple trans-formants. A ubp10� doa1� mutant was constructed by mating a MATa ubp10�(kindly provided by M. Hochstrasser, Yale University) strain with a MAT� doa1�strain; the resulting diploid strain was sporulated and dissected. The MMS andHU sensitivity of the mutants was characterized by growing cultures to mid-logphase, normalizing them by cell density, and serially diluting them in fivefoldincrements. Five-microliter drops were plated on YPD containing different con-centrations of MMS or HU, grown for 2 to 3 days at 30°C, and then photo-graphed. Epitope tagging was performed as previously described (28). Briefly,the genomic copy of POL30 was C terminally tagged with the 3-HA epitope bytransformation of a double-stranded DNA cassette containing the 3-HA hisMX6cassette targeted to replace the stop codon of POL30. The genomic copy ofDOA1 was C terminally tagged with the 13-myc epitope by transformation of adouble-stranded 13-myc kanMX6 cassette targeted to replace the stop codon ofDOA1. The correct insertion of the tags was verified by PCR and by anti-HA oranti-myc immunoblotting.

Ubiquitin complementation. Plasmids YEp96, YEp110 (kindly provided by M.Hochstrasser, Yale University), pES12, and pTER103 (kindly provided by M.Ellison, University of Alberta) were transformed into strains W1588-4C (wildtype) and FR657 (doa1� mutant). Transformants were grown to mid-log phase,normalized by cell density, and serially diluted in fivefold increments. Seven-microliter drops were plated on synthetic complete-Trp media containing differ-ent concentrations of MMS, grown for 2 to 3 days at 30°C, and then photo-graphed. Complementation of the H2B ubiquitination and histone H3 K4methylation was performed by transforming strains BY4741 (wild type) andFR1025 (doa1� mutant) with plasmids YEplac195 and pCUP-Ub-195 (kindlyprovided by E. Johnson, Thomas Jefferson University).

Immunoprecipitation. Cells were grown to mid-log phase, pelleted, washedonce with water, and lysed in buffer A (50 mM Tris, 150 mM NaCl, 5 mM MgCl20.5% Triton X-100, 0.1% deoxycholic acid, 1� Complete mini protease inhibitorcocktail). Lysates were clarified by centrifugation. Total protein (2 mg) wasincubated with either 200 �g bovine serum albumin (BSA; Fisher) or 200 �gubiquitin (Fisher) for 2 h at 4°C. Ubiquitin agarose (25 �l; Boston Biochem) wasthen added, followed by incubation with rocking for 3 h at 4°C. Beads werewashed three times with buffer A, resuspended in sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) loading buffer, and boiled for 5min. Proteins were separated by 10% SDS-PAGE, transferred to polyvinylidenedifluoride (PVDF), detected with anti-myc antibody (1:2,000, 12 h), followed byanti-mouse HRP-conjugated antibody (1:5,000, 1 h), and visualized with ECLPlus (GE Biosciences).

Immunoblotting. For anti-Ub immunoblotting, cells were grown to mid-logphase, pelleted, washed once with water, resuspended in buffer B (50 mM Tris,100 mM NaCl, 0.5% Triton X-100, 0.1% deoxycholic acid, 1� Complete miniprotease inhibitor cocktail), and lysed by mechanical disruption. After concen-tration was normalized by the Bradford assay (4a), proteins were boiled for 5 minin Tricine loading buffer (0.1 M Tris, 24% glycerol, 8% SDS, 0.2 M dithiothreitol,0.02% Coomassie blue G-250). Proteins were separated by 11% SDS-PAGE(Tricine), transferred to PVDF, detected with anti-Ub antibody (1:800, 2 to 3 h)and anti-rabbit HRP-conjugated antibody (1:5,000, 1 h), and visualized with ECLPlus. For histone H2B ubiquitination assays, cells were transformed with Flag-HTB1 and Flag-htb1-K123R plasmids (kindly provided by M. A. Osley, Univer-sity of New Mexico HSC). Cells were grown to early log phase, normalized by celldensity, pelleted, washed once with water, resuspended in SDS-PAGE samplebuffer, and either immediately frozen or boiled for 10 min. For MMS damage,cells were treated with MMS (0.1%) for 30 min. Proteins were separated by 14%SDS-PAGE, transferred to PVDF, detected with anti-Flag antibody (1:2,000,2.5 h) and anti-mouse HRP-conjugated antibody (1:5,000, 1 h), and visualizedwith ECL Plus. For anti-H3 K4 trimethylation immunoblotting, cells were grownto mid-log phase, normalized by cell density, and lysed as previously described(11). Proteins were separated by 16% SDS-PAGE, transferred to PVDF, de-tected with anti-H3 trimethyllysine 4 antibody (1:5,000, 12 h) and anti-rabbitHRP-conjugated antibody (1:5,000, 1 h), and visualized with ECL Plus. Mem-branes used for anti-Flag and anti-H3 trimethyllysine 4 immunoblotting werestripped using Restore Western blotting stripping buffer (Pierce) according tothe manufacturer’s instructions, reprobed with anti-PGK antibody (0.4 �g/ml,12 h) and anti-mouse HRP-conjugated antibody (1:10,000, 1 h), and visualizedwith ECL Plus. For PCNA ubiquitination assays, cells expressing Pol30-3HA

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were grown to mid-log phase, treated with MMS (0.02%, 1 h), pelleted, washedonce with water, and flash frozen. Lysates were prepared by mechanical disrup-tion in buffer B. Equal amounts of proteins were separated by 11% SDS-PAGE,transferred to PVDF, detected with anti-HA antibody (1:1,000, 12 h) and anti-mouse horseradish peroxidase-conjugated antibody (1:10,000, 1 h), and visual-ized with ECL Plus.

Ubiquitin protease assay. BY4741 (wild type) and Doa1-TAP cells were grownto mid-log phase. Lysates were prepared from 1 liter of cells by mechanicaldisruption in lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT,0.5% Triton X-100, 0.1% deoxycholic acid, 1� Complete mini protease inhibitorcocktail). The NaCl concentration of the lysates was increased to 300 mM. Totalprotein (300 mg) was incubated with 100 �l immunoglobulin G (IgG)-Sepharose6 fast flow resin (Amersham) at 4°C for 5 h. The resin was washed five times withlysis buffer containing 300 mM NaCl, two times with lysis buffer containing 500mM NaCl, and three times with ubiquitin protease buffer (50 mM Tris, 5 mMMgCl2, 2 mM DTT). Resin was resuspended in 1 ml of ubiquitin protease buffer.

K48-Ub3 (2 �g; Boston Biochem) was added to a 300-�l aliquot of the resus-pended resin, and the mixture was incubated at 30°C. The reactions werequenched by the addition of Tricine loading buffer and flash frozen. The reac-tions were analyzed by 11% SDS-PAGE (Tricine), transferred to PVDF, de-tected with anti-Ub antibody (1:700, 1 h), followed by anti-rabbit HRP-conju-gated antibody (1:5,000, 1 h), and visualized with ECL Plus.

Real-time PCR. Total RNA from mid-log-phase cultures of strains BY4741(wild type) and FR1025 (doa1� mutant) was prepared using the MasterPureyeast RNA purification kit (Epicentre) according to the manufacturer’s in-structions. RNA (1 �g) was subjected to cDNA synthesis using the iScriptcDNA synthesis kit (Bio-Rad). Quantitative real-time reverse transcription-PCR was carried out on 0.1 �l cDNA in a total reaction volume of 20 �l.Product accumulation was monitored by SYBR green fluorescence. Relativeexpression levels were determined by comparing the RAD6 or POL30 tran-script level to that of the housekeeping gene TCM1. Primer sequences areavailable upon request.

TABLE 1. Yeast strains used in this study

Strain Genotype Source

BY4741 MATa his3�1 leu2�0 met15�0 ura3�0 ATCCorf� BY4741 orf�::kanMX4 Open BiosystemsFR1025 BY4741 doa1�::LEU2 This studyFR381 BY4741 ubi4�::kanMX4 doa1�::LEU2 This studyFR684 BY4741 ubx5�::kanMX4 doa1�::LEU2 This studyFR682 BY4741 ubx6�::kanMX4 doa1�::LEU2 This studyFR683 BY4741 ubx7�::kanMX4 doa1�::LEU2 This studyFR432 BY4741 shp1�::kanMX4 doa1�::LEU2 This studyFR561 BY4741 ubp7�::kanMX4 doa1�::LEU2 This studyFR550 BY4741 rad6�::kanMX4 doa1�::LEU2 This studyFR552 BY4741 rad5�::kanMX4 doa1�::LEU2 This studyFR553 BY4741 rad18�::kanMX4 doa1�::LEU2 This studyFR1021 BY4741 mms2�::kanMX4 doa1�::LEU2 This studyFR1024 BY4741 ubc13�::kanMX4 doa1�::LEU2 This studyFR376 BY4741 srs2�::kanMX4 doa1�::LEU2 This studyFR663 BY4741 bre1�::kanMX4 doa1�::LEU2 This studyFR1026 BY4741 lge1�::kanMX4 doa1�::LEU2 This studyFR685 BY4741 cdc73�::kanMX4 doa1�::LEU2 This studyFR664 BY4741 ubp8�::kanMX4 doa1�::LEU2 This studyFR686 BY4741 htb2�::kanMX4 doa1�::LEU2 This studyFR687 BY4741 hda1�::kanMX4 doa1�::LEU2 This studyFR688 BY4741 htz1�::kanMX4 doa1�::LEU2 This studyFR555 BY4741 rad9�::kanMX4 doa1�::LEU2 This studyFR1218 BY4741 ubp4�::kanMX4 doa1�::LEU2 This studyFR1223 BY4741 rad12�::kanMX4 doa1�::LEU2 This studyFR1225 BY4741 rad14�::kanMX4 doa1�::LEU2 This studyFR521 BY4741 DOA1-13myc This studyMHY501 MATa lys2-801 leu2-3,112 ura3-52 his3-�200 trp1-1 gal2 M. HochstrasserMHY1227 MATa lys2-801 leu2-3,112 ura3-52 his3-�200 trp1-1 ubp10::HIS3 M. HochstrasserFR1231 MHY501 MATa This studyFR1229 MHY501 MATa doa1�::LEU2 This studyFR1232 MHY501 MATa ubp10�::HIS3 This studyFR1230 MHY501 MATa ubp10�::HIS3 doa1�::LEU2 This studyW1588-4C MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 R. RothsteinFR657 W1588-4C doa1�::LEU2 This studySUB280 MATa lys2-801 leu2-3,112 ura3-52 his3-�200 trp1-1 �am ubi1-�1::TRP1

ubi2-�2::ura3 ubi3-�ub-2 ubi4-�2::LEU2 (pUB39, pUB100)D. Finley

FR512 SUB280 doa1�::kanMX6 This studySUB413 MATa lys2-801 leu2-3,112 ura3-52 his3-�200 trp1-1 �am ubi1-�1::TRP1

ubi2-�2::ura3 ubi3-�ub-2 ubi4-�2::LEU2 �Ub K63R �pUB100D. Finley

FR514 SUB413 doa1�::kanMX6 This studyY0003 MATa his3-�200 leu2-3,112 lys2-801 trp1-1 �am ura3-52 S. JentschFR1023 Y0003 doa1�::LEU2 This studyFR1088 Y0003 POL30-3HA This studyFR1090 Y0003 POL30-3HA doa1�::LEU2 This studyY1192 Y0003 pol30-K164R S. JentschFR1022 Y1192 doa1�::LEU2 This studyFR1105 Y1192 pol30-3HA This studyDOA1-TAP BY4741 DOA1-TAP Open BiosystemsEGY48 MATa ura3 trp l his3 6lexAop-LEU2 Origene

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RESULTS

Doa1 interacts with ubiquitin, ubiquitin chains, and ubiq-uitin-like proteins. To identify proteins that may interact withDoa1, a yeast two-hybrid screen was carried out against an S.cerevisiae genomic library using Doa1 fused to LexA as bait. Agenomic library, rather than a cDNA library, was used toensure the representation of genes that might not be expressedunder nondamage conditions. Approximately 1.5 � 106 trans-formants were screened, giving rise to about 200 colonies onselective media. Thirty-nine positive clones were identified bygalactose-dependent growth on Leu-deficient medium andlacZ expression. The library plasmids were recovered and se-quenced. Four proteins were found to interact with Doa1:Ubi4 (29 inserts recovered), Nfi1 (7), Ubx5 (4), and Ubx7 (1).The same screen was performed in the presence of MMS.Positive interactors were selected on galactose-containing,leucine-deficient media supplemented with 0.02% MMS. Allother steps were carried out in a manner identical to that forthe nondamage screen. In the presence of MMS, two proteinswere found to interact with Doa1: Ubi4 (1) and Ubp7 (1).

The most frequently isolated library insert in the yeast two-hybrid screens corresponded to UBI4, which encodes a stress-inducible polyubiquitin chain of five head-to-tail-linked Ubmonomers. All of the isolated clones began at the N terminusof Ubi4 and contained two to four ubiquitins. To examinewhether Doa1 binds Ub monomers and/or polymers, we em-ployed a direct yeast two-hybrid approach to detect interac-

tions between Doa1 and single Ub or Ub chains comprised oftwo to five head-to-tail-linked Ub moieties. Full-length andtruncated constructs were amplified from the genomic UBI4gene, cloned into pJG4-5, and then tested for interaction withDoa1-LexA. We observed that Doa1 binds both Ub monomersand Ub polymers but has a preference for binding polyubiq-uitin chains of two to five Ub moieties (Fig. 1A). These resultsagree with those reported previously by Russell and Wilkinson,who showed that Doa1 interacts with K29-linked polyubiquitinchains (35).

To confirm that Doa1 binds Ub, we incubated Ub agarosewith whole-cell lysate from a DOA1-myc strain in the presenceof either free Ub or BSA. Proteins that bound to the Ubagarose were analyzed by anti-myc Western blotting. Relative

FIG. 1. Doa1 binds Ub and polyubiquitin chains. (A) Direct yeasttwo-hybrid interaction assay between Doa1 and Ub chains. YeastEGY48 was transformed with plasmids expressing Doa1-LexA (DNAbinding domain), Ub1-5 (transcriptional activation domain), and alacZ reporter plasmid, patched onto HC Gal–X-Gal medium, andincubated for 2 days at 30°C. The number of linked Ub monomers, 0to 5, is indicated. (B) Doa1 binds Ub in vitro. Cell lysate (2 mg) derivedfrom strain Doa1-myc (FR521) was incubated in the presence of ubiq-uitin (200 �g) or BSA (200 �g) for 2 h at 4°C. Ubiquitin agarose wasthen added, followed by incubation at 4°C for 3 h. Bound proteins wereseparated by SDS-PAGE and subjected to anti-myc Western blotting.IP, immunoprecipitation.

FIG. 2. DOA1 interacts with the genes encoding the proteins it ispredicted to bind. Fivefold serial dilutions of 105 cells were plated onYPD plates as well as YPD plates containing MMS or HU and incu-bated for 3 days at 30°C. The following strains were used: wild-type(WT) (BY4741), doa1� (FR1025), ubi4�, ubp7�, ubx5�, ubx6�,ubx7�, shp1�, doa1� ubi4� (FR381), doa1� ubp7� (FR561), doa1�ubx5� (FR684), doa1� ubx6� (FR682), doa1� ubx7� (FR683), anddoa1� shp1� (FR432) strains.

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to the BSA control, we observed that free Ub effectively com-peted with the immobilized Ub and significantly reduced thelevel of Doa1 retained on the Ub agarose (Fig. 1B), confirmingthat Doa1 binds Ub.

DOA1 interacts with UBI4 and genes that encode UBX do-mains. Both doa1� and ubi4� mutants are sensitive to heat,canavanine (data not shown), and DNA damage, although thedamage sensitivity of the doa1� mutants is significantly greater(Fig. 2). The similar phenotypes of doa1� and ubi4�, as well asthe observation that doa1� mutants have lower levels of Ub,suggested that the two proteins are at least partially function-ally redundant. To gain more insight into the relationship be-tween the two genes, we examined the DNA damage sensitivityof a doa1� ubi4� mutant. The doa1� ubi4� mutant is syner-gistically more sensitive to both MMS and HU than eithersingle mutant (Fig. 2), confirming that these genes perform atleast partially redundant roles. A similar genetic interactionbetween LUB1 and UBI4 in S. pombe was also observed (30).

Five proteins predicted to bind to Doa1 (Ubx4, Ubx5, Ubx6,Ubx7, and Shp1) contain a UBX domain (7, 12, 43). Althoughthe UBX domain has no significant sequence homology to Ub,it adopts a similar tertiary structure (49). Recently, it wasshown that all seven S. cerevisiae UBX domain-containing pro-teins bind to Cdc48 (which has been shown to bind Doa1 [12])and act to provide substrate specificity (36). We examined thegenetic relationship between DOA1 and genes encoding UBXproteins, including UBX4, UBX5, UBX6, UBX7, and SHP1.Deletion of UBX5, UBX7, or SHP1 suppresses the MMS sen-sitivity of the doa1� mutant, while the ubx6� doa1� mutant issynergistically sensitive to HU (Fig. 2). Deletion of UBX4 hasno effect on the sensitivity of the doa1� mutant. These datasuggest that the UBX proteins form a complex network ofinteractions with Doa1 and that at least part of the function ofDoa1 involves these UBX proteins.

Doa1 is required to process K48-linked Ub trimers. We nextexamined the endogenous levels of Ub in the absence of DOA1(Fig. 3A). As reported previously (14), a reduced level of free

Ub is observed in doa1� mutants. In addition, we also ob-served an accumulation of trimeric Ub (Ub3) in the doa1�mutant. The assignment of the band as Ub3 was based on bothits molecular weight and comparison to a ubp14� mutantwhich is known to accumulate Ub3 (3). In order to determinethe linkage topology of Ub3, the K48R and K63R mutants ofUb were overexpressed in DOA1 and doa1� cells (Fig. 3B).K48R Ub overexpression reduced the level of Ub3 in doa1�cells to that observed in DOA1 cells. However, overexpressionof the K63R mutant did not affect the level of Ub3 in doa1�cells. Thus, it appears that the K48-linked Ub3 (K48-Ub3)accumulates in the absence of Doa1, suggesting that the activ-ity of Doa1 is directly or indirectly related to the consumptionof K48-Ub3.

To further test the hypothesis that Doa1 is involved in thedegradation of K48-Ub3, we immobilized Doa1-TAP fromwhole-cell lysates on IgG-Sepharose beads (Fig. 3C). Afterextensively washing the beads, we added purified K48-Ub3 andincubated the resulting mixture at 30°C. Analysis by Westernblotting with anti-Ub antibody showed a significant accumula-tion of Ub2 and Ub monomer. In contrast, control reactionsusing whole-cell lysates from wild-type cells with untaggedDoa1 showed virtually no degradation of the K48-Ub3. Thisresult suggests that either Doa1 itself is a Ub protease or Doa1copurifies with a Ub protease.

To examine whether the K48-Ub3 protease activity might beprovided by an associated protein, we made and characterizeddouble deletion mutants of DOA1 and genes that encode theknown Ub proteases. Six of the 17 genes that encode Ubproteases showed genetic interactions with DOA1 (Fig. 4).Deletion of UBP7 suppressed the damage sensitivity of thedoa1� mutant (Fig. 2). We also found that the doa1� ubp14�mutant was synergistically more sensitive to MMS and HUthan the single mutants, and the doa1� ubp4� double mutantwas synergistically more sensitive to HU. Deletion of UBP8,UBP10, or UBP12 suppressed the MMS sensitivity of thedoa1� mutant. We next determined whether the deletion of

FIG. 3. Doa1 is required to process K48-linked polyubiquitin trimers (Ub3) into free Ub. (A) The doa1� mutant exhibited low levels of freeUb and accumulation of Ub3. Cell lysates derived from the DOA1 (W1588-4C) and doa1� (FR657) strains were subjected to anti-Ub Westernblotting. (B) Ub3 is K48 linked. Expression of K48R Ub, but not K63R Ub, suppressed the accumulation of Ub3. The DOA1 (W1588-4C) anddoa1� (FR657) strains were transformed with plasmids expressing Ub (YEp96), K48R Ub (YEp110), or K63R Ub (pTER103). Whole-cell lysateswere prepared from mid-log-phase cells treated with CuSO4 (100 �M) for 2 h and subjected to anti-Ub Western blotting. (C) The Doa1 proteincomplex possesses in vitro Ub protease activity. Cell lysates derived from the wild-type (BY4741) and Doa1-TAP strains were incubated withIgG-Sepharose at 4°C for 5 h. After being washed extensively, beads were resuspended and incubated with K48-Ub3 at 30°C. Proteins were resolvedby SDS-PAGE and subjected to anti-Ub Western blotting.

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any of the known Ub protease genes results in accumulation ofUb3, as seen in the doa1� mutant. Anti-Ub Western blotsshowed that in the BY4741 background, only ubp1� andubp14� mutants accumulated Ub3 (see the supplemental ma-terial). We then examined the levels of Ub3 in doa1� ubp1�and doa1� ubp14� mutants. We anticipated that if either Ubp1or Ubp14 provided the Doa1-dependent Ub protease activity,then deletion of the corresponding gene would not increase theaccumulation of Ub3 in doa1� cells. However, in both cases,the double mutants accumulated more Ub3 than either singlemutant (see the supplemental material), suggesting that Doa1-dependent Ub protease activity does not require either UBP1or UBP14, at least not when deleted alone. In addition, be-cause Doa1 and Ubp7 are predicted to physically interact andbecause deletion of UBP7 suppressed the MMS and HU sen-sitivity of the doa1� mutant (Fig. 2), we examined the Ubprofile in the doa1� ubp7� double mutant. Deletion of UBP7did not affect the accumulation of Ub3 in wild-type (see thesupplemental material) or doa1� cells (data not shown). Thesedata suggest that either Doa1 is itself a Ub protease or Doa1-dependent protease activity is provided by one of several dif-ferent Ub proteases that act redundantly.

Doa1 provides Ub for a damage response pathway that uti-lizes K63-linked ubiquitin chains. It was previously demon-strated that overexpression of Ub partially suppresses the Ub-proline–�-galactosidase degradation defect of doa1� cells(14). In addition, Ub expression suppressed the heat sensitivityof S. pombe lub1� cells (30). To examine the effect of Ubexpression on the DNA damage sensitivity of doa1� cells,

exogenous Ub was overexpressed in a doa1� mutant. TheMMS sensitivity associated with doa1� cells was nearly com-pletely complemented by the expression of Ub (Fig. 5A), con-sistent with the idea that Doa1 provides Ub for the DNAdamage response.

We next determined whether Ub-mediated complementa-tion of the doa1� mutant requires K48- or K63-linked Ubpolymers (Fig. 5A). Expression of the K48R Ub mutant sup-pressed the MMS sensitivity of doa1� cells to the same extentas the expression of wild-type Ub. However, expression ofK63R Ub failed to suppress the MMS sensitivity. To furtherinvestigate the relationship of Doa1 and K63-linked Ub chains,we examined the effect of deleting DOA1 in strains engineeredto express the K63R mutant as the sole source of Ub (Fig. 5B).Strains SUB280 and SUB413 carry deletions of all chromo-somal Ub genes (UBI1, UBI2, UBI3, and UBI4) and express Ub(SUB280) or the K63R mutant (SUB413) from a plasmid. Asexpected, the SUB413 strain was significantly more sensitive toMMS than the SUB280 strain; however, the sensitivity wassignificantly suppressed by deletion of DOA1. This suppressionis consistent with a mechanism by which Doa1 degrades K48-

FIG. 4. DOA1 interacts with Ub proteases. Fivefold serial dilutionsof 105 cells were plated on YPD plates as well as YPD plates contain-ing MMS or HU and incubated for 3 days at 30°C. The followingstrains were used: wild-type (WT) (BY4741 except for ubp10 panel,where FR1231 was used), doa1� (FR1025 except for ubp10 panel,where FR1229 was used), ubp4�, ubp10� (FR1232), ubp12�, ubp14�,doa1� ubp4� (FR1218), doa1� ubp10� (FR1230), doa1� ubp12�(FR1223), and doa1� ubp14� (FR1225) strains.

FIG. 5. Doa1 is required to maintain normal levels of K63-linkedUb used in the DNA damage response. (A) Expression of Ub as wellas K48R Ub, but not K63R Ub, suppressed the MMS sensitivity asso-ciated with the doa1� mutant. The DOA1 (W1588-4C) and doa1�(FR657) strains were transformed with plasmids expressing Ub(YEp96), K48R Ub (YEp110), K63R Ub (pTER103), or an emptyvector (pES12). Fivefold serial dilutions of 105 cells were plated onHC-Trp media containing 0.02% MMS. Plates were incubated for 2days at 30°C. (B) Deletion of DOA1 in the SUB413 strain (whichexpresses ubiquitin solely as K63R) suppressed the sensitivity ofSUB413 to MMS. Fivefold serial dilutions of 105 cells were plated onYPD media with or without 0.007% MMS and incubated for 3 days at30°C.

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linked Ub trimers and commits the resulting Ub to a pathwaythat assembles K63-linked polymers. Perhaps, if the K63-linked polymers cannot be produced (i.e., in the SUB413strain), then the activity of Doa1 will result in the nonproduc-tive consumption of the K48-linked polymers, and thus, thedeletion of DOA1 will be beneficial to the cell.

Doa1 supplies Ub for damage-induced PCNA ubiquitina-tion. The involvement of Doa1 in providing Ub for a pathwayinvolving K63-linked Ub chains, as well as the sensitivity ofdoa1� cells to DNA-damaging agents, prompted us to inves-tigate the relationship between DOA1 and the genes that en-code components of the PCNA ubiquitination machinery. TheDNA damage sensitivities of double deletion mutants of DOA1with RAD6, RAD18, RAD5, UBC13, MMS2, and SRS2 weredetermined. All of these double mutants are synergisticallymore sensitive to MMS and HU than the corresponding singlemutants (the genetic interaction between RAD18 and DOA1reported in reference 17 was in error and appeared to resultfrom a suppressor mutation in the rad18� library strain). Inaddition, deletion of DOA1 in a POL30-K164R mutant (inwhich PCNA cannot be ubiquitinated) results in synergisticsensitivity to MMS and HU (Fig. 6). To rule out the possibilitythat these sensitivities result from transcriptional defects in theabsence of DOA1 (see below), we examined the transcriptlevels of RAD6 and POL30 in wild-type and doa1� cells. Tran-script levels for both RAD6 and POL30 were identical in eachcase, suggesting that the genetic interactions reflect disruptionof protein function.

To obtain direct evidence that Doa1 is involved in PCNAubiquitination, the modification of PCNA-HA was monitoredafter DNA damage. The introduction of the HA tag results inincreased sensitivity to DNA damage; however, the sensitivitywas significantly less than that observed in the inactive POL30-K164R mutant (data not shown). After treating cells with MMSto induce ubiquitination of PCNA (21), we observed a slower-migrating species, which we attributed to monoubiquitinatedPCNA-HA (Fig. 7A). Under the same conditions, no PCNAmodification was observed in a POL30-K164R mutant in whichPCNA cannot be ubiquitinated. Ubiquitinated PCNA-HA isalso not observed in the doa1� mutant, suggesting that Doa1 isrequired to ubiquitinate PCNA. Indeed, when the experimentwas repeated in the presence of exogenously expressed Ub, weobserved that PCNA was modified (Fig. 7B). Because we wereunable to detect polyubiquitinated forms of PCNA, we wereunable to address any role Doa1 might have in PCNA poly-ubiquitination. Nonetheless, the data clearly demonstrate thatDoa1 plays a central role in providing Ub for PCNA modifi-cation.

Doa1 is required for histone H2B ubiquitination. A high-throughput synthetic genetic array screen identified a syntheticlethal interaction between DOA1 and CDC73 (42), which is anessential component of the Paf1 complex that is required forH2B ubiquitination (46). We constructed a doa1� cdc73� mu-tant by transforming a haploid cdc73� strain with a deletioncassette targeted for DOA1 and observed a slow growth phe-notype (Fig. 8). The mutant also exhibited synergistic sensitiv-ity to MMS and HU. Based on these results and the geneticinteraction between DOA1 and RAD6, we speculated thatDoa1 might be involved in H2B ubiquitination. We thus de-termined the genetic relationship between DOA1 and other

genes involved in H2B ubiquitination (Fig. 8). Deletion ofDOA1 in bre1� and lge1� mutants resulted in synergistic sen-sitivity to MMS and HU. As mentioned above, deletion ofUBP8 or UBP10, both histone H2B deubiquitinases (2), sup-presses the sensitivity of doa1� cells to DNA damage. More-over, deletion of HTB2, one of the two genes encoding histoneH2B (44), also suppresses the sensitivity of doa1� cells toMMS. These genetic data suggest that Doa1 is involved in theubiquitination of histone H2B.

To determine whether Doa1 is directly involved in H2Bubiquitination, the level of ubiquitinated H2B was examined inthe absence of DOA1. Flag-HTB1 (encoding Flag-tagged H2B)and Flag-htb1-K123R (encoding Flag-tagged H2B that cannotbe ubiquitinated [34]) expression plasmids were transformedinto DOA1 and doa1� strains. Cell lysates were prepared andsubjected to anti-Flag immunoblotting. Deletion of DOA1 re-sults in a decrease in H2B-Ub levels under normal cellularconditions. After treatment with MMS, doa1� cells showed aprofound H2B ubiquitination defect (Fig. 7D). Because MMStreatment decreases the levels of free Ub in DOA1 and doa1�cells to similar extents, this defect is unlikely the result of

FIG. 6. DOA1 interacts with genes involved in PCNA ubiquitina-tion. Fivefold serial dilutions of 105 cells were plated on YPD plates aswell as YPD plates containing MMS or HU and incubated for 3 daysat 30°C. The following strains were used: wild-type (WT) (BY4741),doa1� (FR1025), rad5�, rad18�, mms2�, ubc13�, srs2�, doa1� rad5�(FR552), doa1� rad18� (FR553), doa1� mms2� (FR1021), doa1�ubc13� (FR1024), doa1� srs2� (FR376), wild-type (Y0003), doa1�(FR1023), pol30-K164R (Y1192), and doa1� pol30-K164R (FR1022)strains.

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decreased levels of Ub. Indeed, expression of Ub does notaffect the H2B ubiquitination defect observed in a doa1�strain (Fig. 7E). The data suggest that Doa1 plays a specificrole in histone H2B ubiquitination that is required undernormal cellular conditions and especially in the presence ofDNA damage.

H2B ubiquitination regulates methylation of histone H3 atK4 by Set1. Mutants that have defects in H2B ubiquitinationshow reduced levels of H3 K4 methylation. Likewise, DOA1deletion results in a reduction of H3 methylation at K4 (Fig.7C). Importantly, as with the H2B ubiquitination defect, themethylation defect is not suppressed by Ub overexpression,suggesting that, at least for H2B ubiquitination, Doa1 doesmore than simply produce free cellular Ub. Because this assaydetects methylation of an unmodified and endogenously ex-pressed protein, it also suggests that the observable defects arenot related to ectopic expression of modified H2B. These datasupport the hypothesis that Doa1 plays a specific and function-

ally important role that is required for H2B ubiquitination andthe subsequent H3 K4 methylation.

DOA1 interacts genetically with other genes involved intranscription. The mammalian homolog of Doa1, PLAP, as-sociates with HDAC6 (a class II histone deacetylase), p97/VCP/Cdc48, and polyubiquitin (23, 37). In S. cerevisiae, Doa1has been shown to bind Cdc48 (14), and in this report, wedescribe its binding to polyubiquitin. The homolog of HDAC6is also conserved across species, and its S. cerevisiae homologueHda1 is known to act at the ENA1 promoter and regulate thetranscriptional response to DNA damage (47). We examinedwhether the mammalian PLAP-HDAC6-Cdc48 polyubiquitincomplex is conserved in yeast. Deletion of HDA1 suppressedthe sensitivity of doa1� cells to MMS (Fig. 8). However, effortsto detect a physical interaction between Hda1 and Doa1 byimmunoprecipitation were unsuccessful. It seems likely thatthe genetic interaction reflects a functional interaction, but thephysical interaction is too weak to detect or is disrupted in the

FIG. 7. Doa1-mediated Ub production is required for the ubiquitination or maintenance of monoubiquitinated PCNA and histone H2B andfor the efficient trimethylation of histone H3 on K4. (A) Cell lysates from the DOA1 POL30-3HA (FR1088), doa1� POL30-3HA (FR1090), andpol30-K164R-3HA (FR1105) strains were subjected to anti-HA Western blotting. The addition of MMS (0.02%, 1 h) caused induction of PCNAubiquitination in DOA1. PCNA-Ub conjugates were absent in the doa1� and pol30-K164R strains. (B) Ectopic expression of Ub restored thePCNA ubiquitination defect in doa1� cells. The DOA1 POL30-3HA (FR1088) and doa1� POL30-3HA (FR1090) strains were transformed withplasmids YEplac195 (control) and pCUP-Ub-195 (Ub), grown to mid-log phase, and treated with MMS (0.02%, 1 h). Cell lysates were subjectedto anti-HA Western blotting. (C) The DOA1 (BY4741) and doa1� (FR1025) strains were transformed with plasmid YEplac195 (control) orpCUP-Ub-195 (Ub). Cell lysates were subjected to anti-H3 trimethyllysine 4 Western blotting. Protein loading was verified by reprobing themembranes with anti-PGK antibodies. (D) The DOA1 (BY4741) and doa1� (FR1025) strains were transformed with plasmids expressing eitherFlag-HTB1 or Flag-htb1-K123R, grown to early log phase, and either mock treated or treated with MMS (0.1%, 30 min). Cell lysates were subjectedto anti-Flag Western blotting. (E) The DOA1 (BY4741) and doa1� (FR1025) strains were transformed with plasmids expressing either Flag-HTB1or Flag-htb1-K123R and either YEplac195 (control) or pCUP-Ub-195 (Ub). Cell lysates were subjected to anti-Flag Western blotting. K indicatesFlag-HTB1; R indicates Flag-htb1-K123R. The presence of H2B (H2B) or ubiquitinated H2B (H2B-Ub) is also indicated. Protein loading wasverified by reprobing the membranes with anti-PGK antibodies.

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presence of other proteins. Consistent with this possibility, ithas been reported for mammalian cells that the interactionswithin the PLAP-HDAC6-Cdc48 complex are disrupted by thepresence of Ub (37).

HTZ1 encodes a histone H2A variant, is essential for therecruitment of both RNA Pol II and TATA binding protein to

the GAL1-10 promoters, and is thought to act in a chromatin-remodeling pathway that is partially redundant with H2B ubiq-uitination (25). htz1� cells are not only sensitive to DNA-damaging agents and heat but are also sensitive to thetranscriptional inhibitors 6-azauracil and mycophenolic acid,suggesting that HTZ1 plays a role in transcriptional elongationunder DNA damage and/or stress conditions. Interestingly,doa1� and htz1� cells show synergistic sensitivities to MMSand HU (Fig. 8), suggesting that Doa1 functions in a pathwaythat functionally overlaps with Htz1. We also determined thegenetic interaction of Doa1 with Rad9. In addition to its check-point function, Rad9 is involved in the transcriptional induc-tion of several genes involved in multiple DNA metabolism/repair pathways and is itself regulated by H2B ubiquitination(1, 15). We observed a synergistic sensitivity to MMS in adoa1� rad9� strain (Fig. 8). In all, the data suggest that Doa1is required to maintain appropriate levels of H2B ubiquitina-tion, possibly to help control the transcriptional response toDNA damage.

The doa1� mutant is sensitive to transcriptional inhibitorsand shows a Gal defect. In order to further test the hypothesisthat Doa1 plays a role in transcription, we examined the sen-sitivity of a doa1� mutant to the transcriptional inhibitor6-AU. 6-AU inhibits Pol II elongation and has been used toidentify mutants with defective elongation due to their in-creased sensitivity to 6-AU (38). Consistent with the proposedfunction of Doa1, the doa1� mutant is sensitive to 6-AU (seethe supplemental material). In fact, the level of sensitivity issimilar to that observed for the K123R mutant of H2B, whichcannot be ubiquitinated (48). Histone ubiquitination is re-quired for the proper activation of GAL1 (19, 27). To deter-mine whether Doa1 is involved in transcriptional regulation ofthe GAL locus, the growth of the doa1� mutant was examinedon galactose-containing media. Indeed, a significant growthdefect was observed in doa1� cells relative to wild-type cells(see the supplemental material). In all, the data suggest thatDoa1 is required to ubiquitinate or maintain ubiquitinated H2B.

DISCUSSION

During normal growth, a significant portion of Ub is used totarget proteins for proteasomal degradation, and it is presum-ably sequestered within these pathways. However, in the pres-ence of DNA damage, Ub must quickly be made available forposttranslational modification of proteins involved in sensing,repairing, and/or tolerating the damage, such as PCNA andhistone H2B. Thus, DNA damage might be expected to inducethe expression of UBI4, which has conventionally been thoughtto be the major source of Ub for various stress responses (9).We have found that doa1� and ubi4� mutants share severalphenotypes, including sensitivity to heat (9), canavanine (9),and DNA-damaging agents (17). Moreover, doa1� and ubi4�mutants are synergistically sensitive to MMS and HU, suggest-ing that both proteins might supply Ub for the DNA damageresponse. However, we observed that only the doa1� singlemutant showed strong sensitivity to these damaging agents andthat the deletion of UBI4 results in a significant sensitivity onlyif DOA1 is also absent. In addition, as discussed below, doa1�mutants show no MMS-induced ubiquitination of eitherPCNA or histone H2B, despite the presence of functional

FIG. 8. DOA1 interacts with genes involved in the ubiquitination ormaintenance of ubiquitinated H2B. Fivefold serial dilutions of 105 cellswere plated on YPD plates as well as YPD plates containing MMS orHU and incubated for 3 days at 30°C. The following strains were used:wild-type (WT) (BY4741), doa1� (FR1025), rad6�, bre1�, lge1�,cdc73�, ubp8�, htb2�, hda1�, htz1�, rad9�, doa1� rad6� (FR550),doa1� bre1� (FR663), doa1� lge1� (FR1026), doa1� cdc73� (FR685),doa1� ubp8� (FR664), doa1� htb2� (FR686), doa1� hda1� (FR687),doa1� htz1� (FR688), and doa1� rad9� (FR555) strains.

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UBI4. Thus, we conclude that Doa1 plays the dominant role insupplying Ub for the DNA damage response.

Elements of the DNA damage response that appear to relyon Doa1 include the ubiquitination of both PCNA and histoneH2B. Several observations support the conclusion that Doa1 isinvolved in PCNA ubiquitination. First, DOA1 deletion resultsin synergistic sensitivity to DNA damage when deleted in com-bination with other genes involved in either the monoubiquiti-nation (RAD6 and RAD18) or polyubiquitination (RAD5,UBC13, and MMS2) of PCNA. Second, doa1� mutants showno damage-induced PCNA monoubiquitination. Third, whilewe were unable to visualize polyubiquitinated forms of PCNA,Ub expression suppresses the sensitivity of the doa1� mutantbut only if the expressed Ub is capable of forming the K63-linked polymers required for PCNA polyubiquitination. Fi-nally, ectopic expression of Ub suppressed the PCNA mono-ubiquitination defect. The simplest model consistent with theconclusion is that Doa1 is required to maintain or producesufficient levels of Ub for PCNA modification and possibly forother facets of the damage response.

Several arguments support a more specific function forDoa1 in the ubiquitination of H2B. First, DOA1 interacts withgenes involved in producing or maintaining ubiquitinated H2B(RAD6, BRE1, LGE1, CDC73, UBP8, UBP10, and HTB2) aswell as genes involved in chromatin remodeling or the tran-scriptional response to DNA damage (HTZ1, HDA1, andRAD9). Second, Doa1 is required to maintain ubiquitinatedH2B under normal conditions and especially in response toDNA damage. Third, in the absence of DOA1, histone H3methylation at K4, which is regulated by H2B ubiquitination, issignificantly reduced. Importantly, neither H2B ubiquitinationnor H3 K4 methylation defects are suppressed by ectopic ex-pression of Ub, suggesting that unlike PCNA modification,Doa1 plays a more specific and essential role in the ubiquiti-nation of histone H2B. Finally, the doa1� mutant and mutantsof H2B that cannot be ubiquitinated show similarly impairedtranscription of the GAL1 gene and sensitivity to transcrip-tional inhibitors. It is also interesting to note that deletion ofthe gene that encodes the Doa1 binding partner Shp1 results inMMS sensitivity, and the shp1� mutant was also recentlyshown to confer sensitivity to the transcriptional inhibitor myco-

phenolic acid (8). These data suggest that Doa1 is required forH2B ubiquitination and/or maintenance of the modified pro-tein, as well as the subsequent methylation of histone H3 andthe transcriptional response to DNA damage.

A potential mechanism describing the role of Doa1 is illus-trated in Fig. 9. We suggest that when DNA is damaged, Doa1acts to rapidly recycle Ub from proteosomal degradation path-ways into pathways that modify PCNA and histone H2B andpossibly other proteins. This mechanism is supported by thephysical interaction between Doa1 and Ub polymers, the abil-ity of Doa1, or an associated protein, to catalyze the cleavageof K48-Ub3, and the accumulation in vivo of K48-Ub3 in adoa1� mutant. A functional association between Doa1 and theproteasomal degradation pathways is further supported by thepreviously reported physical interaction between Doa1 andCdc48, which together with Ufd1 and Npl4 recruit substrates tothe 26S proteasome (33). Moreover, the Doa1 binding partnerShp1 has been shown to link the cellular stress response toproteasomal protein degradation (36). While Doa1 may itselfprovide the Ub protease activity, the genetic and physical dataare also consistent with its association with multiple differentUb proteases, such as Ubp7, which may be specific for differentprotein targets, such as histone H2B and PCNA. In addition,specificity may be provided by other factors, such as the Ubxproteins, which may bind Doa1 and act as regulatory subunits.While further elucidation of these details requires additionalexperiments, it is clear that the Doa1-associated pathway is themajor source of Ub for the cellular response to DNA damageand that it also plays an essential role in H2B modification.

Although Doa1 was shown to complement the deletion ofLUB1 in S. pombe, the two genes do not appear to be func-tionally redundant. Lub1 appears to provide Ub specifically forproteasome-mediated protein degradation, whereas we haveshown that Doa1 recycles Ub from proteosomal degradationpathways into pathways that control chromatin structure andreplication. While these apparent contradictions may reflectdifferences between S. cerevisiae and S. pombe, the phenotypesare sufficiently distinct to suggest that despite the high se-quence homology of Doa1 and Lub1 (53% similarity), theirfunctions may have at least partially diverged. Interestingly, themammalian homolog of Doa1, PLAP, has been shown to form

FIG. 9. Model for Doa1 function. The K48-linked Ub trimer, possibly produced as a peptide fusion after proteolytic degradation of ubiquiti-nated proteins, is processed by a complex containing Doa1, and the Ub is channeled into the DNA damage response.

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a complex with HDAC6 and Cdc48, which binds polyubiquitinand copurifies with ubiquitin protease activity (36), suggestingthat the functions of Doa1 might be conserved in S. cerevisiaeand mammals. Further testing of the proposed model for Doa1function, as well as its conservation in other eukaryotes, willgreatly contribute to our understanding of how cells regulateubiquitination and respond to DNA damage.

ACKNOWLEDGMENTS

We gratefully acknowledge M. A. Osley for helpful discussions.Helpful suggestions of a referee are also acknowledged. We thank M.Hochstrasser, M. Ellison, E. Johnson, M. A. Osley, R. Rothstein, D.Finley, and S. Jentsch for providing strains and plasmids.

This work was funded by the NIH (GM068569).

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