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• W2079266876 abstract "The RING finger protein RAD5 interacts and cooperates with the UBC13-MMS2 ubiquitin-conjugating enzyme in postreplication DNA damage repair in yeast. Previous observations implied that the function of UBC13 and MMS2 is dependent on the presence of RAD5, suggesting that the RING finger protein might act as a ubiquitin-protein ligase specific for the UBC13-MMS2 complex. In support of this notion it is shown here that the contact surfaces between the RAD5 RING domain and UBC13 correspond to those found in other pairs of ubiquitin-conjugating enzymes and ubiquitin-protein ligases. Mutations that compromise the protein-protein interactions either between the RING domain and UBC13 or within the UBC13-MMS2 dimer were found to have variable effects on repair activity in vivo that strongly depended on the expression levels of the corresponding mutants. Quantitative analysis of the affinity and kinetics of the UBC13-MMS2 interaction suggests a highly dynamic association model in which compromised mutual interactions result in phenotypic effects only under conditions where protein levels become limiting. Finally, this study demonstrates that beyond its cooperation with the UBC13-MMS2 dimer, RAD5 must have an additional role in DNA damage repair independent of its RING finger domain. The RING finger protein RAD5 interacts and cooperates with the UBC13-MMS2 ubiquitin-conjugating enzyme in postreplication DNA damage repair in yeast. Previous observations implied that the function of UBC13 and MMS2 is dependent on the presence of RAD5, suggesting that the RING finger protein might act as a ubiquitin-protein ligase specific for the UBC13-MMS2 complex. In support of this notion it is shown here that the contact surfaces between the RAD5 RING domain and UBC13 correspond to those found in other pairs of ubiquitin-conjugating enzymes and ubiquitin-protein ligases. Mutations that compromise the protein-protein interactions either between the RING domain and UBC13 or within the UBC13-MMS2 dimer were found to have variable effects on repair activity in vivo that strongly depended on the expression levels of the corresponding mutants. Quantitative analysis of the affinity and kinetics of the UBC13-MMS2 interaction suggests a highly dynamic association model in which compromised mutual interactions result in phenotypic effects only under conditions where protein levels become limiting. Finally, this study demonstrates that beyond its cooperation with the UBC13-MMS2 dimer, RAD5 must have an additional role in DNA damage repair independent of its RING finger domain. ubiquitin-activating enzyme ubiquitin-conjugating enzyme ubiquitin-protein ligase wild type open reading frame glutathione S-transferase resonance units Covalent attachment of ubiquitin to a target protein generates a signal that can function in the regulation of many biological processes, ranging from cell cycle progression and transcriptional activation to inflammatory and immune responses (1Ben-Neriah Y. Nat. Immunol. 2002; 3: 20-26Crossref PubMed Scopus (344) Google Scholar, 2Conaway R.C. Brower C.S. Conaway J.W. Science. 2002; 296: 1254-1258Crossref PubMed Scopus (346) Google Scholar, 3Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6902) Google Scholar, 4Jorgensen P. Tyers M. Curr. Opin. Microbiol. 1999; 2: 610-617Crossref PubMed Scopus (6) Google Scholar). Ubiquitylation of a protein is a multistep reaction involving a complex enzymatic cascade (5Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2920) Google Scholar); in an ATP-dependent reaction, the C terminus of ubiquitin is linked via a thioester bond to a cysteine residue in the active site of ubiquitin-activating enzyme (E1).1 The ubiquitin thioester is then transferred to the active site cysteine of an E2, which mediates the attachment of the ubiquitin C terminus to a lysine residue of the target protein, resulting in an isopeptide bond. This last step is most often catalyzed by another enzyme, termed ubiquitin-protein ligase or E3. Repeated rounds of ubiquitylation result in the formation of long multiubiquitin chains in which each ubiquitin moiety is linked to an internal lysine residue, usually Lys48, of the preceding one (6Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1117) Google Scholar). All of the eukaryotic genomes encode multiple E2s and an even larger number of E3s, the latter being responsible for substrate recognition (5Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2920) Google Scholar, 7Jackson P.K. Eldridge A.G. Freed E. Furstenthal L. Hsu J.Y. Kaiser B.K. Reimann J.D. Trends Cell Biol. 2000; 10: 429-439Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 8Ulrich H.D. Curr. Top. Microbiol. Immunol. 2002; 268: 137-174PubMed Google Scholar). One prominent class of ubiquitin protein ligases is characterized by the presence of a RING domain, a specialized type of zinc finger in which two Zn2+ ions are coordinated by a group of cysteine or histidine residues in a characteristic arrangement (9Borden K.L. Freemont P.S. Huibregtse J.M. Scheffner M. Beaudenon S. Howley P.M. Curr. Opin. Struct. Biol. 1996; 6: 395-401Crossref PubMed Scopus (418) Google Scholar, 10Freemont P.S. Curr. Biol. 2000; 10: R84-R87Abstract Full Text Full Text PDF PubMed Google Scholar, 11Joazeiro C.A. Weissman A.M. Cell. 2000; 102: 549-552Abstract Full Text Full Text PDF PubMed Scopus (1047) Google Scholar). The RING finger has been proposed to fulfill a scaffold function and has in many cases been shown to be involved in E2 binding (12Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (912) Google Scholar, 13Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (943) Google Scholar, 14Xie Y. Varshavsky A. EMBO J. 1999; 18: 6832-6844Crossref PubMed Scopus (143) Google Scholar). The x-ray structure of the mammalian E3 c-Cbl in complex with a cognate E2, UbcH7, has given insight into the molecular details of the RING domain-E2 interaction (15Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar). A similar contact surface was found by NMR analysis in another RING finger E3, CNOT4 (16Albert T.K. Hanzawa H. Legtenberg Y.I. de Ruwe M.J. van den Heuvel F.A. Collart M.A. Boelens R. Timmers H.T. EMBO J. 2002; 21: 355-364Crossref PubMed Scopus (158) Google Scholar, 17Hanzawa H. de Ruwe M.J. Albert T.K. van Der Vliet P.C. Timmers H.T. Boelens R. J. Biol. Chem. 2001; 276: 10185-10190Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Hundreds of RING finger proteins have been described to date; however, although a growing number of them is being identified as ubiquitin ligases (12Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (912) Google Scholar,13Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (943) Google Scholar, 16Albert T.K. Hanzawa H. Legtenberg Y.I. de Ruwe M.J. van den Heuvel F.A. Collart M.A. Boelens R. Timmers H.T. EMBO J. 2002; 21: 355-364Crossref PubMed Scopus (158) Google Scholar, 18Ruffner H. Joazeiro C.A. Hemmati D. Hunter T. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5134-5139Crossref PubMed Scopus (309) Google Scholar), by no means all of them have been shown to possess E3 activity or be involved in ubiquitylation at all, raising the question of what characteristics distinguish ubiquitin ligases from other RING finger proteins. Modification by ubiquitin generally marks a protein for degradation by the 26 S proteasome (3Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6902) Google Scholar). However, ubiquitylation can also convey nonproteolytic signals (2Conaway R.C. Brower C.S. Conaway J.W. Science. 2002; 296: 1254-1258Crossref PubMed Scopus (346) Google Scholar, 19Hicke L. Trends Cell Biol. 1999; 9: 107-112Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 20Pickart C.M. Mol. Cell. 2001; 8: 499-504Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 21Ulrich H.D. Eukaryotic Cell. 2002; 1: 1-10Crossref PubMed Scopus (47) Google Scholar). In particular, nonstandard multiubiquitin chains in which one ubiquitin moiety is linked to the next via Lys63 have been implicated in such diverse processes as DNA damage repair (22Spence J. Sadis S. Haas A.L. Finley D. Mol. Cell. Biol. 1995; 15: 1265-1273Crossref PubMed Google Scholar), endocytosis (23Galan J. Haguenauer-Tsapis R. EMBO J. 1997; 16: 5847-5854Crossref PubMed Scopus (322) Google Scholar), ribosome biogenesis (24Spence J. Gali R.R. Dittmar G. Sherman F. Karin M. Finley D. Cell. 2000; 102: 67-76Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar), mitochondrial inheritance (25Fisk H.A. Yaffe M.P. J. Cell Biol. 1999; 145: 1199-1208Crossref PubMed Scopus (160) Google Scholar), and NFκB signaling (26Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1512) Google Scholar). In yeast and mammals, the only enzymatic activity demonstrated so far to catalyze the assembly of Lys63-linked ubiquitin chains resides in the heterodimeric complex of UBC13 and MMS2, a genuine UBC and a structurally related ubiquitin-conjugating enzyme variant (27Hofmann R.M. Pickart C.M. Cell. 1999; 96: 645-653Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). The function of Lys63-linked ubiquitin chains in DNA damage repair has been attributed to a participation of UBC13 and MMS2 in the postreplication repair pathway (27Hofmann R.M. Pickart C.M. Cell. 1999; 96: 645-653Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 28Broomfield S. Chow B.L. Xiao W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5678-5683Crossref PubMed Scopus (227) Google Scholar), which confers damage tolerance and ensures the completion of genome duplication in situations where lesions in the template strand cause a stalling of the replication machinery (21Ulrich H.D. Eukaryotic Cell. 2002; 1: 1-10Crossref PubMed Scopus (47) Google Scholar, 29Broomfield S. Hryciw T. Xiao W. Mutat. Res. 2001; 486: 167-184Crossref PubMed Scopus (205) Google Scholar, 30Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D.C.1995Google Scholar, 31Lawrence C. BioEssays. 1994; 16: 253-258Crossref PubMed Scopus (147) Google Scholar). The principal mediator of postreplication repair is RAD6 (32Reynolds P. Weber S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 168-172Crossref PubMed Scopus (104) Google Scholar), which encodes another UBC (33Jentsch S. McGrath J.P. Varshavsky A. Nature. 1987; 329: 131-134Crossref PubMed Scopus (546) Google Scholar). It is believed to be targeted to sites of damage by the DNA-binding RING finger protein RAD18 (34Bailly V. Lauder S. Prakash S. Prakash L. J. Biol. Chem. 1997; 272: 23360-23365Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). An error-free subpathway within the RAD6 system is mediated by another chromatin-associated RING finger protein, the SWI/SNF homolog RAD5 (35Johnson R.E. Prakash S. Prakash L. J. Biol. Chem. 1994; 269: 28259-28262Abstract Full Text PDF PubMed Google Scholar). I have previously shown that the function of UBC13 and MMS2 in the RAD6 pathway in yeast is mediated by RAD5, which contacts UBC13 through its RING domain and recruits the E2 heterodimer to the chromatin in response to DNA damage (36Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (352) Google Scholar). Through its association with the RAD6-RAD18 dimer, RAD5 thus coordinates the assembly of two different E2s on damaged chromatin, suggesting a cooperation of RAD6 and the UBC13-MMS2 complex with the RING finger proteins RAD18 and RAD5 in ubiquitin conjugation (36Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (352) Google Scholar). In confirmation of this model it was recently shown that proliferating cell nuclear antigen, a processivity factor for a number of DNA polymerases dedicated to replication as well as repair, is modified by Lys63-linked ubiquitin chains in response to DNA damage (37Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Crossref PubMed Scopus (1737) Google Scholar). This modification depends on the presence of RAD5, UBC13 and MMS2, whereas RAD6 and RAD18 in the absence of the former afford only mono-ubiquitylation (37Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Crossref PubMed Scopus (1737) Google Scholar). Thus, it appears that the UBC13-MMS2-RAD5 assembly indeed functions as a genuine E2-E3 complex for the assembly of Lys63-linked multiubiquitin chains. Support for this notion is presented here by a genetic and biochemical analysis of the protein-protein interactions within this E2-RING finger complex and their consequences for cooperation between RAD5 and the UBC13-MMS2 dimer in DNA damage repair. The wt yeast strain used in this study as well as the isogenic mutantsubc13::HIS3,rad5::HIS3, andubc13::HIS3 rad5::HIS3 have been described previously (36Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (352) Google Scholar). Strain PJ69-4A (38James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar) was used for two-hybrid assays. Standard protocols were followed for the preparation of yeast media and transformations (39Guthrie C. Fink G.R. Guide to Yeast Genetics and Molecular Biology. Academic Press, San Diego, CA1991Google Scholar). SC medium was a modification from that described by Guthrie and Fink (39Guthrie C. Fink G.R. Guide to Yeast Genetics and Molecular Biology. Academic Press, San Diego, CA1991Google Scholar) and contained 100 mg/liter of each amino acid except for leucine, which was present at 200 mg/liter. This corresponds to a cysteine concentration of 0.67 mm. Yeast strains harboring integrative plasmids were propagated in YPD medium following the initial selection on solid and in liquid medium after transformation; strains with centromeric plasmids were maintained in selective SC medium at all times. Two-hybrid constructs bearing the ORFs of RAD5, UBC13, and MMS2 have been described (36Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (352) Google Scholar). Site-directed mutants were generated by polymerase chain reaction and recloned into the two-hybrid vectors, and the amplified regions were fully sequenced. The intron was removed during construction of the UBC13 mutants; however, its presence or absence did not affect protein levels. 2H. D. Ulrich, unpublished observation. For expression under the control of the native promoter, the RAD5 ORF and the RING finger mutants were recloned from the two-hybrid vectors asBamHI/PstI fragments into a derivative of the integrative vector YIplac211 (40Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2521) Google Scholar) carrying anEcoRI/BamHI fragment encompassing 245 bp of theRAD5 upstream region and a PstI/SphI fragment derived from pGBT9 (Clontech) as a transcriptional terminator (YIp211-PRAD5-RAD5).UBC13 and its mutants were expressed under the control of the native promoter in a similar vector in which 996 bp of theUBC13 upstream regions were inserted as anEcoRI/BamHI fragment upstream of the ORF (YIp211-PUBC13-UBC13). Regulatable expression levels were achieved by placing the UBC13 wt or mutant ORFs under the control of the MET3 promoter into the centromeric vector YCplac111 (40Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2521) Google Scholar), again in combination with the pGBT9-derived transcriptional terminator (YCp111-PMET3-UBC13). For the production of recombinant proteins in Escherichia coli, UBC13 and MMS2 as well as the respective mutants were recloned into expression vectors, allowing the production of different fusion proteins. pGEX-4T-1 (AmershamBiosciences) was used to produce N-terminal GST fusions of UBC13 and its mutants, MMS2, and GST alone. pQE-30 (Qiagen) served as an expression vector for His6-tagged UBC13. The MMS2 ORF as well as the mutant F8A were inserted into the vector pTYB12 (New England Biolabs). This vector affords expression of MMS2 as an N-terminal fusion to a chitin-binding domain, linked by the self-cleavable VMA1 intein sequence, which allows single-step purification of MMS2 bearing three additional amino acids (AGH) at its N terminus. For simplicity, this construct will be referred to as MMS2 in the following text. Sequence maps of all the constructs used in this study are available on request. Analysis of the interactions between RAD5, UBC13, and MMS2 in the two-hybrid system was performed as described previously, using growth on histidine-selective medium as an indication for a positive interaction (36Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (352) Google Scholar). UV sensitivities were determined by means of survival curves or gradient plate assays as described previously (36Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (352) Google Scholar, 41Ulrich H.D. Nucleic Acids Res. 2001; 29: 3487-3494Crossref PubMed Scopus (53) Google Scholar). All of the irradiations were performed at 1.67 J m−2 s−1. Selective medium was used before and after irradiation for strains bearing centromeric vectors. Yeast cultures were grown at 28 °C to exponential phase. Samples of 108cells were lysed by NaOH/β-mercaptoethanol treatment, and the total protein was precipitated with trichloroacetic acid as described (42Silver P.A. Chiang A. Sadler I. Genes Dev. 1988; 2: 707-717Crossref PubMed Scopus (48) Google Scholar). Aliquots were analyzed on 15% SDS-polyacrylamide gels, followed by Western blots using an affinity-purified anti-UBC13 antibody (see below). Recombinant proteins were produced in E. coli BL21(DE3) harboring the respective expression constructs by induction with 1 mmisopropyl-β-d-thiogalactopyranoside for 6 h at 28 °C. Purifications were achieved in a single step by affinity chromatography on the appropriate columns (glutathione-Sepharose (Amersham Biosciences), nickel-nitrilotriacetic acid resin (Novagen or Qiagen), or chitin beads (New England Biolabs) for GST, His6, or chitin-binding domain-intein fusions, respectively) according to the manufacturers' recommendations. Purified proteins were dialyzed against 25 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.5 mm EDTA and stored frozen at −80 °C in the presence of 10% (v/v) glycerol. The protein concentrations were determined by absorbance at 280 nm based on calculated extinction coefficients. Polyclonal antibodies against the His6-UBC13 protein were raised in rabbit and purified by affinity chromatography on CH Sepharose 4B (Amersham Biosciences) covalently derivatized with GST-UBC13. Interaction between UBC13 and MMS2 was analyzed by surface plasmon resonance using a Biacore X instrument. The proteins were dialyzed against HBS buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 3 mmEDTA, 0.05% P-20 detergent). A CM5 chip was derivatized with 15,000 RU of an anti-GST antibody (Biacore) by amine coupling according to the manufacturer's recommendation. Subsequently, ∼850 RU of the GST fusion protein (ligand) were immobilized in one of the flow cells. A reference surface was generated by loading of ∼500 RU of underivatized GST in the other flow cell. The signal in this reference cell was subtracted online during all measurements. The soluble binding partner (analyte) was injected at a range of concentrations in 2-min pulses at a flow rate of 5 μl/min. Because of the fast dissociation rates, no regeneration of the chip surface was necessary between individual injections. After analysis of one particular set of proteins, the ligand was removed from the surface by a 2-min pulse of 10 mm glycine, pH 2.2, followed by immobilization of the next GST fusion protein to be analyzed. The signal at equilibrium (RU eq) was plotted against the analyte concentration C, and the steady state affinity K Aand dissociation constant K D were determined by a fit of this plot according to the model RU eq =K A·C·RU max/(1 + K A·C), whereRU max is the theoretical binding capacity at infinite analyte concentration and K D = 1/K A. Following the BiaEvaluationTMprogram, the fits were considered acceptable if the χ2values were below 10 and the residuals did not exceed a few RU. Ubiquitin chain synthesis was assayed at 30 °C in 25 mm Tris-HCl, pH 7.5, 50 mm NaCl, 10 mm MgCl2, 10 mm ATP, and 10 mm dithiothreitol. Yeast E1 (Affiniti Research) was used at a concentration of 70 nm. Bovine ubiquitin (Sigma) was present at 0.5 mg/ml (58 μm). The reactions were set up with equimolar amounts of GST-UBC13 and MMS2 (0.5 or 5.0 μm each). The samples were withdrawn after 0, 1, 2, and 4 h, and the aliquots were analyzed by SDS-PAGE and Western blots, using an anti-ubiquitin antiserum (Sigma). Site-directed mutagenesis of UBC13 and the RAD5 RING domain was guided by structural information on the UBC13-MMS2 dimer (43VanDemark A.P. Hofmann R.M. Tsui C. Pickart C.M. Wolberger C. Cell. 2001; 105: 711-720Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) as well as two mammalian RING finger ubiquitin-protein ligases in complex with their cognate E2s, c-Cbl with UbcH7 (15Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar) and CNOT4 with UbcH5 (16Albert T.K. Hanzawa H. Legtenberg Y.I. de Ruwe M.J. van den Heuvel F.A. Collart M.A. Boelens R. Timmers H.T. EMBO J. 2002; 21: 355-364Crossref PubMed Scopus (158) Google Scholar). Sequence alignment of the RAD5 RING finger with the corresponding domains of c-Cbl and CNOT4 (Fig.1 A) revealed sufficient homology to allow the design of RAD5 mutations predicted to influence the contact with UBC13. Mutation C914S, which affects one of the cysteines involved in Zn2+ coordination, had previously been shown to disrupt interaction with the E2 (36Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (352) Google Scholar). Because this change is likely to disturb the integrity of the entire domain, additional more subtle mutations were designed by changing predicted surface residues to alanine. Residue Ile916 is conserved in many RING domains, and the corresponding amino acids in both c-Cbl and CNOT4 contact the respective E2. Positions Glu943 and Tyr944 could be aligned with the interface residues Ser407 and Trp408 in c-Cbl, and Asn959 again corresponds to a position involved in E2 contacts in both c-Cbl and CNOT4. In addition, D923A was designed as a control mutation representing a hydrophilic, thus likely surface-exposed residue distant to the putative E2 interface. In the c-Cbl-UbcH7 complex the E2 contacts the RING finger and adjacent regions by means of charged amino acids within its N-terminal α-helix (H1) as well as residues in the loops between β-sheets S3 and S4 as well as α-helices H2 and H3 (Ref. 15Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar and Fig. 1 B). The same structural elements are used in the binding of UbcH7 to a HECT type E3 (44Huang L. Kinnucan E. Wang G. Beaudenon S. Howley P.M. Huibregtse J.M. Pavletich N.P. Science. 1999; 286: 1321-1326Crossref PubMed Scopus (439) Google Scholar). The crystal structures of the yeast and the human UBC13-MMS2 dimers revealed that the corresponding residues in UBC13 are accessible, and the presence of MMS2 would permit simultaneous contacts to the RAD5 RING finger (43VanDemark A.P. Hofmann R.M. Tsui C. Pickart C.M. Wolberger C. Cell. 2001; 105: 711-720Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 45Moraes T.F. Edwards R.A. McKenna S. Pastushok L. Xiao W. Glover J.N. Ellison M.J. Nat. Struct. Biol. 2001; 8: 669-673Crossref PubMed Scopus (138) Google Scholar). Therefore, mutations in UBC13 were designed at the following positions: K6E and K10E in helix H1 and M64A and S96A in the loops. Mutation of Pro97, although more closely representative of the respective contact site in UbcH7 than Ser96, was avoided to exclude a perturbation of the overall protein structure. Mutation E55A was introduced as a control, because this residue had previously been shown to reside in the interface to MMS2 (43VanDemark A.P. Hofmann R.M. Tsui C. Pickart C.M. Wolberger C. Cell. 2001; 105: 711-720Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 45Moraes T.F. Edwards R.A. McKenna S. Pastushok L. Xiao W. Glover J.N. Ellison M.J. Nat. Struct. Biol. 2001; 8: 669-673Crossref PubMed Scopus (138) Google Scholar) and should therefore have no effect on interaction with the RAD5 RING finger. The mutants designed within the RAD5 RING finger were analyzed with respect to their interactions with UBC13 in the two-hybrid system. This method has been used previously to demonstrate the interaction between the two proteins and correlates well with results obtained by co-immunoprecipitation (36Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (352) Google Scholar). To exclude the possibility that a negative result might be due to improper folding or expression of the mutant protein in the two-hybrid system, dimerization with RAD5 itself, which is independent of the RAD5-UBC13 contact, was assayed in parallel. Fig. 2 Ashows that all of the mutants retained association with RAD5 itself; however, they varied considerably in their ability to interact with UBC13. Mutation C914S, an exchange likely to disrupt the structural integrity of the entire RING domain, had previously been shown to prevent the interaction with UBC13 (36Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (352) Google Scholar). Mutation D923A had no effect on association with UBC13, which was expected because this residue should occupy a position in the RING finger facing away from the putative E2 contact site. Mutations I916A, Y944A, and N959A, all predicted to reside on the surface of the RING domain facing the E2, indeed abolished interaction with UBC13 in the two-hybrid system. In contrast, mutation of Glu943, also predicted to contribute to the E2 interface, to alanine did not prevent interaction with UBC13. Thus, the interaction of the RAD5 RING finger with UBC13 involves a surface similar but not identical to that within other RING domains, including several highly conserved residues but also showing variations that are likely to affect the respective E2 specificity. In the reciprocal approach, the ability of the putative contact site mutants of UBC13 to interact with RAD5 was examined. Interaction with MMS2, which is independent of the UBC13-RAD5 association (36Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (352) Google Scholar), was examined in parallel to ensure proper function of the mutants in the two-hybrid system. UBC13(E55A) served as a control for a protein that should only be affected in its association with MMS2 but not with RAD5. The results of the two-hybrid analysis are shown in Fig. 2 B. As expected, mutant UBC13(E55A) was found to associate with RAD5, but interaction with MMS2 was abolished. In contrast, all other mutants were capable of association with MMS2 but had lost their ability to interact with RAD5. These results indicate that amino acids critical for UBC13-RAD5 contacts reside within the N-terminal helix of UBC13 (Lys6 and Lys10) as well as the loops L1 (Met64) and L2 (Ser96) (Fig. 1 B). Thus, the UBC13 interface to the RAD5 RING finger closely resembles the corresponding interfaces of other E2 enzymes with their respective ubiquitin-protein ligases. To examine the effects of the different contact site mutations on the ability of RAD5 and UBC13 to cooperate in DNA damage repair in vivo, the mutant genes were integrated into the genome and expressed under the control of their own promoters in the respective deletion strains. Fig. 3 shows a comparison of the UV sensitivities of representative mutants. Mutant C914S, in which one of the Zn2+-coordinating cysteines is replaced by serine, showed by far the largest effect on cell survival; in fact, its UV sensitivity was more severe than that of theubc13 deletion, indicating that a structural perturbation of the RING domain affects more aspects of RAD5 function than solely its cooperation with UBC13 (Fig. 3 A). Nevertheless, C914S was epistatic to ubc13, because deletion of the E2 in this mutant had no additional effect on UV sensitivity, suggesting that UBC13 function in DNA repair entirely depends on the integrity of the RAD5 RING domain. Yet, this mutant was less sensitive to UV irradiation than a rad5 deletion; thus, RAD5 must have an additional role in DNA repair that is independent of its RING finger. Mutant I916A displayed a UV sensitivity very similar to that of the ubc13deletion (Fig. 3 A). The fact that the double mutantubc13 rad5(I916A) showed no further increase in UV sensitivity indicates that mutation of this residue apparently eliminates all aspects of RAD5 functions that involve a cooperation with UBC13 but leave all of its UBC13-independent functions intact. Mutation D923A had no influence on the UV sensitivity of the respective strain, again confirming that this residue i" @default.
• W2079266876 created "2016-06-24" @default.
• W2079266876 creator A5077688118 @default.
• W2079266876 date "2003-02-01" @default.
• W2079266876 modified "2023-10-11" @default.
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