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Molecular cloning and functional characterization of two murine cDNAs which encode Ubc variants involved in DNA repair and mutagenesis
Biochimica et Biophysica Acta 1519 (2001) 70^77
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Molecular cloning and functional characterization of two murine cDNAs which encode Ubc variants involved in DNA repair and mutagenesis Janelle Franko, Carolyn Ashley, Wei Xiao * Department of Microbiology and Immunology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, Canada S7N 5E5 Received 10 January 2001 ; received in revised form 29 March 2001; accepted 29 March 2001
Abstract Ubiquitin-conjugating enzyme (Ubc) variants share structural similarity with Ubcs but lack the essential cysteine residue required to form a thioester bond with ubiquitin. Yeast Mms2 is a Ubc variant and plays an important role in error-free DNA postreplication repair to protect cells from killing by DNA damaging agents and mutagenesis. Ironically, one of two known Mms2 homologs, CROC1, has been linked to cell immortalization and tumorigenesis. To further investigate cellular roles played by mammalian Mms2 homologs, we report here the molecular cloning, tissue distribution and functional characterization of two mouse cDNAs encoding mMMS2 and mCROC1. Unlike human CROC1, the mCROC1 gene does not encode two alternative transcripts in most tissues. Instead, nonoverlapping sequences were found in two distinct cDNA clones that together would constitute a full-length open reading frame homologous to CROC1B. Both mMMS2 and the C-terminal mCROC1 core domain are able to complement the yeast mms2 mutant functionally and are able to interact with Ubc13 in a yeast two-hybrid assay, indicating that they are true yeast Mms2 homologs and may play a similar role in DNA postreplication repair. We propose several hypotheses to reconcile the seemingly contradictory observations regarding roles of the two mammalian Mms2 homologs in tumorigenesis and carcinogenesis. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Ubc variant; CROC1; MMS2; DNA postreplication repair ; Protein interaction ; Murine
1. Introduction Ubiquitination is a biochemical process found in all eukaryotes and is required for many cellular functions including DNA repair, cell-cycle regulation, control of gene expression and translation [1]. Altered ubiquitination activity has been linked to many human diseases including cancer [2,3]. The complexity of ubiquitination has been increased with the recent discovery of ubiquitin (Ub) variants [4] and Ub-conjugating enzyme (Ubc) variants (UEVs) [5^9] in organisms from yeast to humans. UEVs are characterized by a central domain that has signi¢cant similarity to true Ubcs. However, unlike typical Ubcs, UEVs lack the essential Cys residue and hence the catalytic activity required to form a thiol-ester with Ub in the second step of ubiquitination. Abbreviations : Ub, ubiquitin; Ubc, ubiquitin-conjugating enzyme; UEV, Ubc variant; MMS, methylmethane sulfonate; ORF, open reading frame * Corresponding author. Fax: +1-306-966-4311; E-mail : [email protected]
Saccharomyces cerevisiae MMS2 encodes the ¢rst UEV that has been identi¢ed and genetically characterized. MMS2 was found to be a member of the RAD6 DNA postreplication repair (PRR) and mutagenesis pathway [5]. Unlike most other members within this pathway, the mms2 mutation elevated the spontaneous mutation rate in a REV3-dependent manner, and is synergistic with the rev3 mutation with respect to killing by a wide range of DNA damaging agents such UV, Q-rays and methylmethane sulfonate (MMS). These observations led to a proposal that MMS2 is exclusively involved in the error-free branch of PRR that is parallel to the REV3 mutagenesis subpathway [5,10]. Hence, MMS2 acts to protect yeast cells from endogenous as well as environmentally induced DNA damage and mutagenesis. Recently, Mms2 was found to form a complex with Ubc13 and the heterodimer is required for a novel K63-G76 polyubiquitin chain assembly in vitro [11], suggesting that Mms2 is a positive regulator of Ubc13 activity. Indeed, ubc13 is epistatic to mms2 [11] and, like MMS2, UBC13 also belongs to the error-free PRR pathway [12]. Two human MMS2 homologs (CROC1 and hMMS2)
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have been identi¢ed and characterized by searching human cDNA libraries [9]. CROC1 (contingent replication of cDNA-1) was originally isolated in a screen to identify cDNAs that transactivate the c-fos promoter [7]. It was subsequently isolated through mRNA di¡erential display and found to be upregulated during virus-induced cellular immortalization [6] and downregulated when HT29-M6 human colon carcinoma cells underwent di¡erentiation [8]. In addition, CROC1 appears to be overexpressed in all tumor cell lines examined [6]. In contrast, hMMS2 has relatively low transcript levels in normal tissue and is overexpressed in some but not all human cancer cell lines [9]. hMMS2 and CROC1 share greater than 90% amino acid identity in their core domains, although CROC1 possesses an additional N-terminal domain with two di¡erential splicing products (CROC1A and CROC1B). Both hMMS2 and CROC1 are able to complement the yeast mms2 null mutant with respect to its sensitivity to DNA damaging agents; however, deletion of the CROC1 N-terminal domain is required for complementation of the yeast mutant [9]. Although these observations de¢ne a novel UEV family at both structural and functional levels, there are many questions concerning the biological functions of CROC1 and hMMS2 with regard to tumorigenesis and carcinogenesis. First, several independent observations [6^8] point to CROC1 being a candidate proto-oncogene. It would be of great interest to see if overexpression or deletion of CROC1 a¡ects the potential for tumorigenesis and carcinogenesis. Second, it is ironic that while yeast MMS2 plays a role in error-free PRR to limit spontaneous and damage-induced mutagenesis, at least one of its human homologs (CROC1) is implicated in promoting tumorigenesis [6^9,13]. In addition, unlike CROC1, hMMS2 has not been assigned any possible cellular functions. Does it play a role in error-free PRR like its yeast counterpart ? What are the genetic interactions between MMS2 and CROC1 if both of their products form complexes with UBC13? What are the consequences when CROC1 or hMMS2 is mutated? In order to address all of the above questions, it is necessary to establish an experimental animal model. To this end, we report here the isolation and characterization of two distinct mouse cDNAs homologous to CROC1 and hMMS2. Our results suggest that these murine homologs likely are representatives of their human counterparts. 2. Materials and methods 2.1. Screen of murine cDNA libraries Two mouse VYES cDNA libraries, D10 and V13, were constructed and provided by Dr. S. Elledge (Baylor College of Medicine, Houston, TX) and ampli¢ed as instructed (S. Elledge, personal communication). Standard phage V library screening protocol was followed with
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MMS2 and CROC1B speci¢c probes prepared as follows. A human hMMS2 fragment was generated by EcoRI digestion of the cDNA clone resulting in the ORF plus 100 bp of the 3P untranslated region [9]. The human CROC1B probe is a 2.1 kb fragment containing the entire CROC1B cDNA [7]. Positive clones after tertiary screen were used to infect bacterial strain BNN132 for automatic subcloning [14]. The resulting plasmids were isolated and nucleotide sequences of the inserts were determined by combined manual sequencing using a T7 DNA sequencing kit (Phamacia) and the automatic sequencing facility in the Saskatoon Cancer Center. 2.2. Northern hybridization A mouse multiple tissue Northern membrane was purchased from Clontech (Palo Alto, CA) and used according to the manufacturer's instructions. hMMS2 and CROC1 probes were the same as those used in cDNA library screening. The mouse CROC1 N- and C-terminal speci¢c probes were made by polymerase chain reaction (PCR) ampli¢cation of the mouse cDNA coding regions. The Lactin probe was provided by Clontech as an internal control. 2.3. Plasmid construction Plasmid V13-4 contains mouse MMS2 (mMMS2) cDNA but lacks the ¢rst ¢ve codons including the translation start as judged by comparison with a mouse genomic mMMS2 pseudogene sequence that had previously been isolated (unpublished results). To create a full-length mMMS2 cDNA, oligonucleotide mMMS2-11 (5P-CCGGATCCATGGCAGTCTCCACAGGAGTTAAAGTTCCTCGTAATTTTCGC-3P) containing these ¢ve codons (underlined) and a BamHI site 5P to the translation start codon was used as a primer and V13-4 cDNA as a template for PCR. The resulting PCR product was cloned into the BamHI and EcoRI sites of pTZ19R (Phamacia) to form pTZ-mMMS2, and the entire insert was sequenced. The BamHI^EcoRI fragment was further subcloned into the yeast expression vector pYES2.0 (2 Wm, URA3, PGAL1 TCYC1 , Invitrogen) to form pYES-mMMS2. The unique BamHI site in pTZ-mMMS2 was converted to EcoRI and the resulting EcoRI fragment containing mMMS2 was cloned into a constitutive expression vector YEp126 (2 Wm, URA3, PADH1 -TCYC1 ) in correct orientation to form YEp-mMMS2. In order to express the coding sequence found in clone D10-9, oligonucleotide mCROC1-7 (5P-CGGAATTCATGGTAAAAGTCCCTCGAAATTTC-3P) was designed to create a BamHI site followed by a translation start codon (underlined) at the 5P end of the cDNA insert sequence. The mCROC1 ORF was PCR ampli¢ed using mCROC1-7 and a 3P speci¢c primer and cloned into the BamHI^EcoRI sites of pYES2.0 to form pYES-mCROC1.
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To construct plasmids for the two-hybrid analysis, mCROC1 and mMMS2 coding regions without a stop codon were PCR ampli¢ed, isolated as HindIII^SalI and BamHI^SalI fragments, and cloned into the corresponding sites of pG4BD-0 (from Dr. R.M. Brazas, University of California, San Francisco, CA) as C-terminal fusions to form pmCROC1-G4BD and pmMMS2-G4BD, respectively. The yeast UBC13 coding region was PCR ampli¢ed and isolated as a BamHI-SalI fragment and cloned into pGAD424 (Clontech) as an N-terminal fusion to form pGAD-UBC13. All three fusion constructs were tested as functional in complementing the corresponding yeast mutants (data not shown). 2.4. Yeast experiments Yeast cells were cultured at 30³C in a rich (YPD) or a selective medium containing synthetic dextrose (SD) and the required amino acids and bases [15]. Transformation of yeast strains was carried out using a modi¢ed DMSO transformation protocol [16]. Galactose media was prepared by replacing 2% glucose in the YPD media with 2% D(+)-galactose. Haploid S. cerevisiae strain FY86 (MAT K his3-v200 ura3-52 leu2-v1 GAL ) and its mms2: :LEU2 derivative FY86M2L were used for killing experiments as previously described [5], and DBY747 (MATa his3-1, leu2-3, 112 trp1-289 ura3-52) and its mms2: :LEU2 derivative SBL containing a revertible trp1-289 amber mutation were used for spontaneous mutagenesis assays as described [5]. The Trp reversion rates were calculated using the modi¢ed Luria and Delbruck £uctuation test [17]. DBY747, SBL and their corresponding rev3v mutants WXY382 and WXY665 [10] were also used for killing experiments. 2.5. Two-hybrid analysis The S. cerevisiae strain Y190 (MATa gal4 gal80 his3 trp1 ade2-101 ura3 leu2: :URA3 GAL1-LacZ: :Lys2: :
GAL1-HIS3, received from Dr. D. Gietz (University of Manitoba, Canada) was cotransformed with two plasmids containing pG4BD (TRP1, Gal4BD ) and pGAD424 (LEU2, Gal4AD ) or their derivatives and selected on SD medium lacking both Trp and Leu. Individual transformants were restreaked and then subjected to a ¢lter assay to determine the L-galactosidase (L-gal) activity [18]. Brie£y, 5^10 independent double transformants with both Gal4BD and Gal4AD fusion constructs were grown on SD selective medium for 2 days. Cells were transferred to Whatman No. 1 ¢lter paper, immersed in liquid nitrogen for 10 s to permeabilize cells, and placed on top of another ¢lter which was presoaked in a mixture of 1.8 ml Z-bu¡er containing 5 Wl L-mercaptoethanol and 45 Wl of 20 mg/ml Xgal (5-bromo-4-chloro-3-indolyl-L-D-galactoside) in N,Ndimethylformamide. Plates were sealed with para¢lm and incubated at 30³C. Color development was recorded after a 4-h incubation. 3. Results 3.1. Murine MMS2 and CROC1 cDNAs Screening of the two mouse VYES cDNA libraries V13 and D10 revealed a nearly complete ORF in clone V13-4 that was highly conserved with hMMS2. This clone appeared to lack the ¢rst 15 nucleotides of the ORF based on information from an hMMS2 pseudogene that had been previously isolated (data not shown). After the ¢rst 15 nucleotides were restored to the cDNA clone, the resulting mouse ORF encoded a 145-amino-acid protein that was 97.9% (142/145) identical to hMMS2 and approximately 50% identical to yeast Mms2 (Fig. 1). The same libraries were also screened for sequences homologous to CROC1 using CROC1B cDNA as a probe. Two positive clones, named D10-3 and D10-9, were isolated and their inserts were essentially identical, with D109 being a few nucleotides longer than D10-3 at the 5P end
Fig. 1. Amino acid sequence alignment of S. cerevisiae Mms2 (GenBank accession no. U66724), mMMS2 (accession no. AF303828), hMMS2 (accession no. AF049140), mCROC1 (accession no. AF303829) and hCROC1B (accession no. U39361). The report was generated by a Clustal method with the DNASTAR MegAlign program. It should be noticed that the mCROC1 sequence was assembled from two separate cDNA clones. The C-terminal portion derived from clone D10-9 starts at Val-85 (marked by *).
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of the coding region. Sequence analysis showed that the two clones were highly similar to CROC1B at the nucleotide sequence level. However, they do not appear to contain a full-length mCROC1 coding region, since they lack homology with the N-terminus of CROC1A or CROC1B, and lack a translation start codon. D10-9 contains 137 codons with 90.5% (124/137) sequence identity to C-terminal CROC1 and 84.7% (116/137) identity to mMMS2 (Fig. 1). Rapid ampli¢cation of cDNA ends (RACE) failed to extend further the 5P end of the D10-9 insert. Initial search of the mouse EST database using as a query the CROC1A and CROC1B 5P sequences missing in clone D10-9 revealed two EST clones. One of these clones was isolated by the Washington University^NHMI Mouse EST project from the Stratagene mouse skin cDNA library (#937313). We obtained this EST clone from the ATCC and determined the nucleotide sequence of the entire cDNA insert. The clone could encode 84 amino acids sharing 92.9% (78/ 84) sequence identity with the corresponding CROC1B Nterminus. It does not overlap with D10-9, but instead together they form a continuous sequence to create a complete ORF highly homologous to CROC1B without any nucleotide gaps (Fig. 1). A further search of mouse CROC1 homologs in the EST database using full-length CROC1B as the query showed that out of 40 putative mCROC1 ESTs homologous to the D10-9 insert, 63% (25/40) have 5P ends identical in length to D10-9, 88% (35/40) are within a few nucleotides, whereas the rest are even shorter. Fourteen ESTs were homologous to the above ATCC EST. Of these, seven had the same 3P end, while the other seven were shorter than the ATCC EST. Hence, there is yet no evidence that the 5P and 3P mouse cDNAs homologous to CROC1B are physically linked. 3.2. Tissue distribution of mMMS2 and mCROC1 A mouse multiple tissue Northern membrane was hybridized with CROC1B and hMMS2 to gain insight into the transcript size, the number of transcripts and tissue speci¢city of these UEVs. Although hMMS2 and CROC1 are greater than 90% identical within their core domains, their cDNA probes do not cross-hybridize [9] and therefore the same membrane can be used to compare their transcripts. hMMS2 shares 95% nucleotide sequence identity with mMMS2 in the coding region and hybridized to a major 1.4 kb mouse transcript (Fig. 2A) plus a few minor transcripts of 2.0, 3.4 and 4.0 kb, with an additional 1.0 kb major transcript displayed in the testis. This is in contrast to human tissues where only a 1.5 kb transcript was observed from all tissues examined, including testis ([9], and data not shown). Unlike human CROC1, which has two distinct transcripts, CROC1A (2.0 kb) and CROC1B (2.1 kb) [6,7], two distinct mouse CROC1 transcripts could only be found in kidney (2.4 and 2.0 kb) and testis (2.0 and 1.5 kb). All other tissues displayed only a
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Fig. 2. Expression of mMMS2 and mCROC1 in di¡erent mouse tissues. The membrane containing multiple tissue poly(A) RNA (2 Wg per lane) was sequentially hybridized with (A) the hMMS2 probe, (B) the CROC1B probe, and (C) the L-actin probe. Probe was stripped from the membrane before the next round of hybridization as instructed by the membrane supplier.
single 2.0 kb transcript (Fig. 2B). Both probes showed strong hybridization to mouse heart, brain liver, skeletal muscle, kidney and testis while the spleen expressed extremely low levels of both transcripts (Fig. 2). Overall, the relative tissue distribution and transcript size of mMMS2 and mCROC1 were rather di¡erent from their human homologs. Human CROC1B shares 86% nucleotide sequence identity with the coding region of D10-9 cDNA, and 87% nucleotide sequence identity with that of the putative N-terminal mCROC1 coding region. Hence, the CROC1B probe likely recognizes both mRNA sequences. To further address whether or not both CROC1B homologous sequences exist in a single mouse mRNA molecule, we performed Northern hybridization as described above using N- and C-terminal-speci¢c mouse cDNA probes. To our surprise, the C-terminal cDNA probe (from D10-9) only
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Fig. 3. mMMS2 protects mms2 cells from killing by DNA damaging agents. Yeast strains FY86 (F), FY86mms2 (8) and FY86mms2 transformed with either vector pYES2.0 (a) or pYES-mMMS2 (O) were treated with (A) UV at di¡erent doses or (B) 0.3% MMS for the given times. (C) Yeast strains DBY747 (F, WT), SBL (8, mms2), WXY384 (W, rev3), WXY665 (R, mms2 rev3) and WXY665 transformed with either vector YEp126 (a) or YEpmMMS2 (b) were treated with 0.2% MMS for the given times. Percent survival was scored after a 3-day incubation on YPD plates. The results are the average of at least three independent experiments with standard deviations shown as error bars.
hybridized with the 2.0 kb mRNA in all tissues examined, whereas the N-terminal cDNA probe only recognized the 2.4 kb band in kidney and the 1.5 kb band in testis (data not shown). Hence, this ¢nding further strengthens our previous speculation that the 5P and 3P mouse CROC1B homologous sequences are not physically linked. 3.3. mMMS2 and mCROC1 complement the yeast mms2 null mutant As a ¢rst step in addressing the roles of UEVs in the mouse, we wished to determine if the two cloned mouse MMS2 homologs are able to complement the corresponding yeast mutant. Three di¡erent assays, which had previously been used to characterize the mms2 mutant [5], were carried out. The mms2 null mutant is sensitive to DNA damaging agents such as MMS and UV. The mouse MMS2 ORF was cloned behind an inducible GAL1 promoter to provide high level expression in a Gal yeast mutant strain. Results presented in Fig. 3 demonstrate that mMMS2 fully restored the yeast mms2 mutant to the wild-type level of resistance to killing by UV (Fig. 3A) and MMS (Fig. 3B). However, the yeast mms2 mutant did not display a severe sensitivity to MMS and UV, which is attributed to the functional mutagenesis pathway represented by a nonessential Rev3 DNA polymerase j backing up the loss of the MMS2 error-free PRR pathway [5,10]. Yeast cells carrying both rev3 and mms2 mutations become extremely sensitive to DNA damaging agents, due to simultaneous inactivation of both the error-free and error-prone PRR pathways [5,10]. To demonstrate that mMMS2 functions in the error-free PRR pathway, a mms2 rev3 double mutant was created and used as a recipient to constitutively express mMMS2. Results from MMS killing experiments (Fig. 3C) show that mMMS2 rescued the mms2 rev3 double mutant from killing by
MMS to a level comparable to the rev3 single mutant. These results indicate that (i) mMMS2 indeed functions in the error-free PRR pathway in yeast ; (ii) mMMS2 can functionally replace yeast MMS2; and (iii) high level constitutive expression of mMMS2 does not alleviate defects due to loss of other DNA damage tolerance pathway(s). Finally, one of the most surprising e¡ects of the mms2 mutation is its enhanced spontaneous mutation rate compared with wild-type cells. In this experiment, the mms2 mutant displayed a greater than 50-fold increase in spontaneous Trp reversion rate. Expression of mMMS2 in this strain signi¢cantly reduced the mutation rate (Table 1). A few fold increase above the wild-type level was probably due to loss of the YEp-mMMS2 in a small portion of transformants during nonselective growth. Like mMMS2, mCROC1 isolated from D10-9 was also able to rescue the yeast mms2 mutant from killing by MMS, as shown in Fig. 4. It is interesting to note that mCROC1 used in this experiment is similar in length and sequence to the arti¢cially truncated CROC1B(v80) in a previous study [9].
Table 1 Saccharomyces cerevisiae spontaneous mutation rates Straina
Mutation rate (U1038 )b
Relative ratec
DBY747 (WT) SBL (mms2) SBL/YEp-mMMS2
1.49 þ 1.32 76.5 þ 4.2 6.2 þ 4.0
1 51.3 4.2
a All strains were derived from wild-type DBY747, which carries the revertible trp1-289 amber mutation. b The spontaneous mutation rate is expressed as the number of revertants per cell per generation. The results are an average of three separate experiments with standard deviations. c Relative to the wild-type rate.
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Fig. 4. Protection of mms2 cells by mCROC1 from MMS-induced killing. Yeast strains FY86 (lane 1), FY86mms2 (lane 2), FY86mms2/pYES-mCROC1 (lane 3) and FY86mms2/pYES2.0 (lane 4) were imprinted onto gradient plates containing either YPGal or YPGal+0.03% MMS and the plates were incubated for 42 h at 30³C before photographing. The arrow points towards the higher MMS concentration.
3.4. mMMS2 and mCROC1 interact with Ubc13 It has been reported recently that Ubc13 catalyzes a unique Lys63 polyUb chain assembly and that this activity requires its in vitro physical association with Mms2 [11]. To see if cloned mouse UEVs also function by forming a heterodimer with Ubc13, we performed an in vivo twohybrid analysis (Fig. 5). Yeast cells carrying mMMS2,
mCROC1 or UBC13 alone fused to Gal4DB or Gal4AD coding sequences were unable to transactivate the endogenous PGAL -lacZ reporter gene. In contrast, if a Y190 cell simultaneously carried both Gal4BD -mMMS2 (or Gal4BD mCROC1) and Gal4AD -UBC13, it expressed strong L-gal activity indicative of stable mMMS2 (or mCROC1)-Ubc13 complex formation in vivo. This result shows that both mMMS2 and the truncated mCROC1 are able to interact
Fig. 5. Interaction of mCROC1 and mMMS2 with Ubc13 by a two-hybrid ¢lter assay. S. cerevisiae strain Y190 was cotransformed with pG4BDmCROC1/pGAD-UBC13, pG4BD-mMMS2/pGAD-UBC13, pG4BD-mCROC1/pGAD424, pG4BD-mMMS2/pGAD424, or pG4BD-0/pGAD-UBC13. At least ¢ve independent colonies from each transformation were analyzed and two representative colonies are shown. The positive control was Y190 transformed with pCL2 (YCp-GAL4) and the negative control was Y190 cotransformed with pG4BD-0/pGAD424. The ¢lter was incubated in the Xgal solution at 30³C for 4 h.
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with Ubc13 and probably act as positive regulators of Ubc13 activity. 4. Discussion Genes encoding Ubc variants have been discovered only recently and form at least two families. One family, represented by TSG101, was isolated through screening of mouse tumor suppressor genes [19] and its human homolog has been linked to cancers [20,21]. TSG101 family proteins contain a domain with signi¢cant homology to Ubcs and it has been postulated that they are regulatory proteins in the ubiquitination pathway [22,23]. The second UEV family, represented by Mms2 [5], consists of small proteins in which the entire sequence forms a tertiary structure highly resembling a Ubc core domain (M. Ellison, W. Xiao, unpublished results). Given that the yeast Mms2 is a positive regulator of Ubc13 in a unique polyubiquitination process [11], functions in the error-free PRR pathway and limits spontaneous and damage-induced mutagenesis [5], it is surprising that CROC1, one of its human homologs, was isolated from several laboratories as a putative proto-oncogene [6^8]. To reconcile the aforementioned two seemingly con£icting observations, we propose two alternative hypotheses. The ¢rst hypothesis is that although Ubc13/Mms2 homologs from di¡erent species share signi¢cant homology and perform similar biochemical reactions, their target molecules are di¡erent in human and yeast cells. While the Ubc13-Mms2 targets in yeast cells are PRR proteins, those in human cells may be involved in cellular processes such as cell cycle progression and tumorigenesis. Indeed, it was recently shown that while yeast Ubc13 interacts with a DNA repair RING ¢nger domain protein Rad5 [24], human UBC13 and CROC1/hMMS2 are required for the RING ¢nger protein TRAF6-mediated activation of IUB kinase [13]. The second hypothesis is that CROC1 and hMMS2 may confer di¡erent functions. While CROC1 is a putative proto-oncogene, hMMS2 may be exclusively involved in DNA repair. This hypothesis is consistent with our recent observation that suppression of hMMS2 in human cells by antisense RNA results in phenotypes characteristic of error-free PRR defects (Z. Li, V. Maher, W. Xiao, unpublished results). Although evidence to date favors the above hypotheses, we do not rule out other possibilities. For example, CROC1 and hMMS2 may be multifunctional in a manner reminiscent of the human major DNA repair AP endonuclease, APE/HAP1 [25], also identi¢ed as Ref1, a bifunctional protein capable of stimulating sequencespeci¢c AP-1 DNA binding activity [26]. As an initial step towards understanding the biological and tumorigenic roles of mammalian MMS2 homologs in a whole animal model, we have reported here the molecular cloning of both mCROC1 and mMMS2 cDNAs and characterized their functions in yeast cells. Several issues
raised from this study are discussed below. First of all, we have shown that as in human cells, the mouse has two distinct Mms2 homologs with a very high degree of amino acid identity but signi¢cant nucleotide divergence from each other. This observation indicates the existence of two distinct subfamilies within the MMS2 gene family and that each of the subfamilies may have unique biological functions. It also suggests that the mouse is probably a suitable model for characterizing the MMS2 gene family and its roles in human diseases. Secondly, a few di¡erences have been noticed in the expression of the mouse and human MMS2 homologs, particularly with respect to CROC1. These include lack of alternative CROC1 transcripts in all mouse tissues examined, as well as di¡erences in tissue distribution of both genes in human and mouse. Lastly, probably the most surprising observation is that all mCROC1 cDNAs so far isolated from di¡erent tissues and cell cultures are truncated at the site separating the Mms2homologous core domain and the N-terminal coding sequences. cDNAs encoding both sequences have been identi¢ed that would form a continuous CROC1B homolog; however, there is no evidence that they are physically linked in a single mRNA molecule. Furthermore, mouse mRNAs from di¡erent tissues exclusively hybridize with either N-terminal or C-terminal-speci¢c probes, but not both. The cDNA encoding the core domain mCROC1 lacks a translation start, but is able to complement the corresponding yeast mms2 mutant when supplemented with a translation start codon. A few scenarios may be considered to explain these observations. (i) The mCROC1 5P and 3P coding sequences are in two di¡erent exons that may be joined rarely, ine¤ciently, only in some tissues, or only under particular environmental stress (e.g., DNA damage). (ii) These two cDNAs, although both are homologous to di¡erent regions of CROC1B, are from two distinct mouse genes. This scenario is less likely since the 5P coding cDNA does not have a stop codon and the 3P coding cDNA does not have a start codon; hence, we have to assume further that both cDNAs represent stand-alone exons that have to be further processed or joined to other transcripts. (iii) Given our previous observation that the N-terminally truncated, but not the full length CROC1B, can replace yeast MMS2 functions [9], it is possible that the N-terminal mCROC1 coding sequence is a regulatory element of mCROC1 activity and that the truncated cDNAs correspond to post-transcriptional modi¢cation of the full-length mCROC1. It is further proposed that this post-transcriptional regulation of mCROC1 activity may be replaced in human cells by alternative splicing that yields two distinct CROC1 transcripts. In view of this hypothesis, it is interesting to note that CROC1A is reported to be functional in yeast [27] while CROC1B is not [9]. Recent studies [24,28] have revealed that the yeast PRR and mutagenesis pathway involves at least two Ubc (Ubc2/Rad6 and Ubc13-Mms2) and two RING-¢nger do-
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main proteins (Rad5 and Rad18) and that these proteins interact physically and genetically. So far only one transgenic mouse model (HR6B) has been reported [29]. However, this model has not been informative with respect to PRR, probably due to the second mouse Rad6 homolog HR6A [30]. Molecular cloning of mMMS2 and mCROC1 genomic DNA in conjunction with the mouse genome project may shed light on this issue. Hence, creation of transgenic mice de¢cient in mMMS2 and/or mCROC1 will address how these genes are involved in diverse cellular processes including cell cycle progression, di¡erentiation, immortalization and DNA postreplication repair. Acknowledgements The authors wish to thank Dr. S. Elledge for mouse cDNA libraries and other laboratories for reagents. This work was supported by Medical Research Council of Canada operating grant MT-15076 to W.X. J.F. was supported by a University of Saskatchewan College of Medicine Graduate Fellowship and C.A. was supported by a University of Saskatchewan College of Medicine Postdoctoral Fellowship. References [1] A. Ciechanover, The ubiquitin-proteasome proteolytic pathway, Cell 79 (1994) 13^21. [2] A. Alves-Rodrigues, L. Gregori, M.E. Figueiredo-Pereira, Ubiquitin, cellular inclusions and their role in neurodegeneration, Trends Neurosci. 21 (1998) 516^520. [3] A. Ciechanover, A. Orian, A.L. Schwartz, The ubiquitin-mediated proteolytic pathway : mode of action and clinical implications, J. Cell. Biochem. 34 (Suppl.) (2000) 40^51. [4] E.T. Yeh, L. Gong, T. Kamitani, Ubiquitin-like proteins: new wines in new bottles, Gene 248 (2000) 1^14. [5] S. Broom¢eld, B.L. Chow, W. Xiao, MMS2, encoding a ubiquitinconjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway, Proc. Natl. Acad. Sci. USA 95 (1998) 5678^5683. [6] L. Ma, S. Broom¢eld, C. Lavery, S.L. Lin, W. Xiao, S. Bacchetti, Upregulation of CIR1/CROC1 expression upon cell immortalization and in tumor-derived human cell lines, Oncogene 17 (1998) 1321^1326. [7] M.L. Rothofsky, S.L. Lin, CROC-1 encodes a protein which mediates transcriptional activation of the human FOS promoter, Gene 195 (1997) 141^149. [8] E. Sancho, M.R. Vila, L. Sanchez-Pulido, J.J. Lozano, R. Paciucci, M. Nadal, M. Fox, C. Harvey, B. Bercovich, N. Loukili, A. Ciechanover, S.L. Lin, F. Sanz, X. Estivill, A. Valencia, T.M. Thomson, Role of UEV-1, an inactive variant of the E2 ubiquitin-conjugating enzymes, in in vitro di¡erentiation and cell cycle behavior of HT-29M6 intestinal mucosecretory cells, Mol. Cell. Biol. 18 (1998) 576^589. [9] W. Xiao, S.L. Lin, S. Broom¢eld, B.L. Chow, Y.F. Wei, The products of the yeast MMS2 and two human homologs (hMMS2 and CROC-1) de¢ne a structurally and functionally conserved Ubc-like protein family, Nucleic Acids Res. 26 (1998) 3908^3914. [10] W. Xiao, B.L. Chow, T. Fontanie, L. Ma, S. Bacchetti, T. Hryciw, S. Broom¢eld, Genetic interactions between error-prone and error-free postreplication repair pathways in Saccharomyces cerevisiae, Mutat. Res. 435 (1999) 1^11.
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[11] R.M. Hofmann, C.M. Pickart, Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair, Cell 96 (1999) 645^653. [12] J. Brusky, Y. Zhu, W. Xiao, UBC13, a DNA-damage-inducible gene, is a member of the error-free postreplication repair pathway in Saccharomyces cerevisiae, Curr. Genet. 37 (2000) 168^174. [13] L. Deng, C. Wang, E. Spencer, L. Yang, A. Braun, J. You, C. Slaughter, C. Pickart, Z.J. Chen, Activation of the IUB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain, Cell 103 (2000) 351^361. [14] S.J. Elledge, J.T. Mulligan, S.W. Ramer, M. Spottswood, R.W. Davis, VYES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations, Proc. Natl. Acad. Sci. USA 88 (1991) 1731^1735. [15] F. Sherman, G.R. Fink, J. Hicks, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1983. [16] J. Hill, K.A. Donald, D.E. Gri¤ths, G. Donald, DMSO-enhanced whole cell yeast transformation, Nucleic Acids Res. 19 (1991) 5791. [17] M.S. Williamson, J.C. Game, S. Fogel, Meiotic gene conversion mutants in Saccharomyces cerevisiae. I. Isolation and characterization of pms1-1 and pms1-2, Genetics 110 (1985) 609^646. [18] P.L. Bartel, S. Fields, Analyzing protein-protein interactions using two-hybrid system, Methods Enzymol. 254 (1995) 241^263. [19] L. Li, S.N. Cohen, Tsg101 : a novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells, Cell 85 (1996) 319^329. [20] L. Li, X. Li, U. Francke, S.N. Cohen, The TSG101 tumor susceptibility gene is located in chromosome 11 band p15 and is mutated in human breast cancer, Cell 88 (1997) 143^154. [21] Y. Oh, M.L. Proctor, Y.H. Fan, L.K. Su, W.K. Hong, K.M. Fong, Y.S. Sekido, A.F. Gazdar, J.D. Minna, L. Mao, TSG101 is not mutated in lung cancer but a shortened transcript is frequently expressed in small cell lung cancer, Oncogene 17 (1998) 1141^1148. [22] C.P. Ponting, Y.D. Cai, P. Bork, The breast cancer gene product TSG101 : a regulator of ubiquitination ?, J. Mol. Med. 75 (1997) 467^469. [23] E.V. Koonin, R.A. Abagyan, TSG101 may be the prototype of a class of dominant negative ubiquitin regulators, Nat. Genet. 16 (1997) 330^331. [24] H.D. Ulrich, S. Jentsch, Two RING ¢nger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair, EMBO J. 19 (2000) 3388^3397. [25] B. Demple, T. Herman, D.S. Chen, Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: de¢nition of a family of DNA repair enzymes, Proc. Natl. Acad. Sci. USA 88 (1991) 11450^11454. [26] S. Xanthoudakis, T. Curran, Identi¢cation and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity, EMBO J. 11 (1992) 653^665. [27] T.M. Thomson, H. Khalid, J.J. Lozano, E. Sancho, J. Arino, Role of UEV-1A, a homologue of the tumor suppressor protein TSG101, in protection from DNA damage, FEBS Lett. 423 (1998) 49^52. [28] W. Xiao, B.L. Chow, S. Broom¢eld, M. Hanna, The Saccharomyces cerevisiae RAD6 group is composed of an error-prone and two errorfree postreplication repair pathways, Genetics 155 (2000) 1633^ 1641. [29] H.P. Roest, J. van Klaveren, J. de Wit, C.G. van Gurp, M.H. Koken, M. Vermey, J.H. van Roijen, J.W. Hoogerbrugge, J.T. Vreeburg, W.M. Baarends, D. Bootsma, J.A. Grootegoed, J.H. Hoeijmakers, Inactivation of the HR6B ubiquitin-conjugating DNA repair enzyme in mice causes male sterility associated with chromatin modi¢cation, Cell 86 (1996) 799^810. [30] M.H. Koken, E.M. Smit, I. Jaspers-Dekker, B.A. Oostra, A. Hagemeijer, D. Bootsma, J.H. Hoeijmakers, Localization of two human homologs, HHR6A and HHR6B, of the yeast DNA repair gene RAD6 to chromosomes Xq24-q25 and 5q23-q31, Genomics 12 (1992) 447^453.
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Molecular cloning and functional characterization of two murine cDNAs which encode Ubc variants involved in DNA repair and mutagenesis Janelle Franko, Carolyn Ashley, Wei Xiao * Department of Microbiology and Immunology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, Canada S7N 5E5 Received 10 January 2001 ; received in revised form 29 March 2001; accepted 29 March 2001
Abstract Ubiquitin-conjugating enzyme (Ubc) variants share structural similarity with Ubcs but lack the essential cysteine residue required to form a thioester bond with ubiquitin. Yeast Mms2 is a Ubc variant and plays an important role in error-free DNA postreplication repair to protect cells from killing by DNA damaging agents and mutagenesis. Ironically, one of two known Mms2 homologs, CROC1, has been linked to cell immortalization and tumorigenesis. To further investigate cellular roles played by mammalian Mms2 homologs, we report here the molecular cloning, tissue distribution and functional characterization of two mouse cDNAs encoding mMMS2 and mCROC1. Unlike human CROC1, the mCROC1 gene does not encode two alternative transcripts in most tissues. Instead, nonoverlapping sequences were found in two distinct cDNA clones that together would constitute a full-length open reading frame homologous to CROC1B. Both mMMS2 and the C-terminal mCROC1 core domain are able to complement the yeast mms2 mutant functionally and are able to interact with Ubc13 in a yeast two-hybrid assay, indicating that they are true yeast Mms2 homologs and may play a similar role in DNA postreplication repair. We propose several hypotheses to reconcile the seemingly contradictory observations regarding roles of the two mammalian Mms2 homologs in tumorigenesis and carcinogenesis. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Ubc variant; CROC1; MMS2; DNA postreplication repair ; Protein interaction ; Murine
1. Introduction Ubiquitination is a biochemical process found in all eukaryotes and is required for many cellular functions including DNA repair, cell-cycle regulation, control of gene expression and translation [1]. Altered ubiquitination activity has been linked to many human diseases including cancer [2,3]. The complexity of ubiquitination has been increased with the recent discovery of ubiquitin (Ub) variants [4] and Ub-conjugating enzyme (Ubc) variants (UEVs) [5^9] in organisms from yeast to humans. UEVs are characterized by a central domain that has signi¢cant similarity to true Ubcs. However, unlike typical Ubcs, UEVs lack the essential Cys residue and hence the catalytic activity required to form a thiol-ester with Ub in the second step of ubiquitination. Abbreviations : Ub, ubiquitin; Ubc, ubiquitin-conjugating enzyme; UEV, Ubc variant; MMS, methylmethane sulfonate; ORF, open reading frame * Corresponding author. Fax: +1-306-966-4311; E-mail : [email protected]
Saccharomyces cerevisiae MMS2 encodes the ¢rst UEV that has been identi¢ed and genetically characterized. MMS2 was found to be a member of the RAD6 DNA postreplication repair (PRR) and mutagenesis pathway [5]. Unlike most other members within this pathway, the mms2 mutation elevated the spontaneous mutation rate in a REV3-dependent manner, and is synergistic with the rev3 mutation with respect to killing by a wide range of DNA damaging agents such UV, Q-rays and methylmethane sulfonate (MMS). These observations led to a proposal that MMS2 is exclusively involved in the error-free branch of PRR that is parallel to the REV3 mutagenesis subpathway [5,10]. Hence, MMS2 acts to protect yeast cells from endogenous as well as environmentally induced DNA damage and mutagenesis. Recently, Mms2 was found to form a complex with Ubc13 and the heterodimer is required for a novel K63-G76 polyubiquitin chain assembly in vitro [11], suggesting that Mms2 is a positive regulator of Ubc13 activity. Indeed, ubc13 is epistatic to mms2 [11] and, like MMS2, UBC13 also belongs to the error-free PRR pathway [12]. Two human MMS2 homologs (CROC1 and hMMS2)
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have been identi¢ed and characterized by searching human cDNA libraries [9]. CROC1 (contingent replication of cDNA-1) was originally isolated in a screen to identify cDNAs that transactivate the c-fos promoter [7]. It was subsequently isolated through mRNA di¡erential display and found to be upregulated during virus-induced cellular immortalization [6] and downregulated when HT29-M6 human colon carcinoma cells underwent di¡erentiation [8]. In addition, CROC1 appears to be overexpressed in all tumor cell lines examined [6]. In contrast, hMMS2 has relatively low transcript levels in normal tissue and is overexpressed in some but not all human cancer cell lines [9]. hMMS2 and CROC1 share greater than 90% amino acid identity in their core domains, although CROC1 possesses an additional N-terminal domain with two di¡erential splicing products (CROC1A and CROC1B). Both hMMS2 and CROC1 are able to complement the yeast mms2 null mutant with respect to its sensitivity to DNA damaging agents; however, deletion of the CROC1 N-terminal domain is required for complementation of the yeast mutant [9]. Although these observations de¢ne a novel UEV family at both structural and functional levels, there are many questions concerning the biological functions of CROC1 and hMMS2 with regard to tumorigenesis and carcinogenesis. First, several independent observations [6^8] point to CROC1 being a candidate proto-oncogene. It would be of great interest to see if overexpression or deletion of CROC1 a¡ects the potential for tumorigenesis and carcinogenesis. Second, it is ironic that while yeast MMS2 plays a role in error-free PRR to limit spontaneous and damage-induced mutagenesis, at least one of its human homologs (CROC1) is implicated in promoting tumorigenesis [6^9,13]. In addition, unlike CROC1, hMMS2 has not been assigned any possible cellular functions. Does it play a role in error-free PRR like its yeast counterpart ? What are the genetic interactions between MMS2 and CROC1 if both of their products form complexes with UBC13? What are the consequences when CROC1 or hMMS2 is mutated? In order to address all of the above questions, it is necessary to establish an experimental animal model. To this end, we report here the isolation and characterization of two distinct mouse cDNAs homologous to CROC1 and hMMS2. Our results suggest that these murine homologs likely are representatives of their human counterparts. 2. Materials and methods 2.1. Screen of murine cDNA libraries Two mouse VYES cDNA libraries, D10 and V13, were constructed and provided by Dr. S. Elledge (Baylor College of Medicine, Houston, TX) and ampli¢ed as instructed (S. Elledge, personal communication). Standard phage V library screening protocol was followed with
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MMS2 and CROC1B speci¢c probes prepared as follows. A human hMMS2 fragment was generated by EcoRI digestion of the cDNA clone resulting in the ORF plus 100 bp of the 3P untranslated region [9]. The human CROC1B probe is a 2.1 kb fragment containing the entire CROC1B cDNA [7]. Positive clones after tertiary screen were used to infect bacterial strain BNN132 for automatic subcloning [14]. The resulting plasmids were isolated and nucleotide sequences of the inserts were determined by combined manual sequencing using a T7 DNA sequencing kit (Phamacia) and the automatic sequencing facility in the Saskatoon Cancer Center. 2.2. Northern hybridization A mouse multiple tissue Northern membrane was purchased from Clontech (Palo Alto, CA) and used according to the manufacturer's instructions. hMMS2 and CROC1 probes were the same as those used in cDNA library screening. The mouse CROC1 N- and C-terminal speci¢c probes were made by polymerase chain reaction (PCR) ampli¢cation of the mouse cDNA coding regions. The Lactin probe was provided by Clontech as an internal control. 2.3. Plasmid construction Plasmid V13-4 contains mouse MMS2 (mMMS2) cDNA but lacks the ¢rst ¢ve codons including the translation start as judged by comparison with a mouse genomic mMMS2 pseudogene sequence that had previously been isolated (unpublished results). To create a full-length mMMS2 cDNA, oligonucleotide mMMS2-11 (5P-CCGGATCCATGGCAGTCTCCACAGGAGTTAAAGTTCCTCGTAATTTTCGC-3P) containing these ¢ve codons (underlined) and a BamHI site 5P to the translation start codon was used as a primer and V13-4 cDNA as a template for PCR. The resulting PCR product was cloned into the BamHI and EcoRI sites of pTZ19R (Phamacia) to form pTZ-mMMS2, and the entire insert was sequenced. The BamHI^EcoRI fragment was further subcloned into the yeast expression vector pYES2.0 (2 Wm, URA3, PGAL1 TCYC1 , Invitrogen) to form pYES-mMMS2. The unique BamHI site in pTZ-mMMS2 was converted to EcoRI and the resulting EcoRI fragment containing mMMS2 was cloned into a constitutive expression vector YEp126 (2 Wm, URA3, PADH1 -TCYC1 ) in correct orientation to form YEp-mMMS2. In order to express the coding sequence found in clone D10-9, oligonucleotide mCROC1-7 (5P-CGGAATTCATGGTAAAAGTCCCTCGAAATTTC-3P) was designed to create a BamHI site followed by a translation start codon (underlined) at the 5P end of the cDNA insert sequence. The mCROC1 ORF was PCR ampli¢ed using mCROC1-7 and a 3P speci¢c primer and cloned into the BamHI^EcoRI sites of pYES2.0 to form pYES-mCROC1.
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To construct plasmids for the two-hybrid analysis, mCROC1 and mMMS2 coding regions without a stop codon were PCR ampli¢ed, isolated as HindIII^SalI and BamHI^SalI fragments, and cloned into the corresponding sites of pG4BD-0 (from Dr. R.M. Brazas, University of California, San Francisco, CA) as C-terminal fusions to form pmCROC1-G4BD and pmMMS2-G4BD, respectively. The yeast UBC13 coding region was PCR ampli¢ed and isolated as a BamHI-SalI fragment and cloned into pGAD424 (Clontech) as an N-terminal fusion to form pGAD-UBC13. All three fusion constructs were tested as functional in complementing the corresponding yeast mutants (data not shown). 2.4. Yeast experiments Yeast cells were cultured at 30³C in a rich (YPD) or a selective medium containing synthetic dextrose (SD) and the required amino acids and bases [15]. Transformation of yeast strains was carried out using a modi¢ed DMSO transformation protocol [16]. Galactose media was prepared by replacing 2% glucose in the YPD media with 2% D(+)-galactose. Haploid S. cerevisiae strain FY86 (MAT K his3-v200 ura3-52 leu2-v1 GAL ) and its mms2: :LEU2 derivative FY86M2L were used for killing experiments as previously described [5], and DBY747 (MATa his3-1, leu2-3, 112 trp1-289 ura3-52) and its mms2: :LEU2 derivative SBL containing a revertible trp1-289 amber mutation were used for spontaneous mutagenesis assays as described [5]. The Trp reversion rates were calculated using the modi¢ed Luria and Delbruck £uctuation test [17]. DBY747, SBL and their corresponding rev3v mutants WXY382 and WXY665 [10] were also used for killing experiments. 2.5. Two-hybrid analysis The S. cerevisiae strain Y190 (MATa gal4 gal80 his3 trp1 ade2-101 ura3 leu2: :URA3 GAL1-LacZ: :Lys2: :
GAL1-HIS3, received from Dr. D. Gietz (University of Manitoba, Canada) was cotransformed with two plasmids containing pG4BD (TRP1, Gal4BD ) and pGAD424 (LEU2, Gal4AD ) or their derivatives and selected on SD medium lacking both Trp and Leu. Individual transformants were restreaked and then subjected to a ¢lter assay to determine the L-galactosidase (L-gal) activity [18]. Brie£y, 5^10 independent double transformants with both Gal4BD and Gal4AD fusion constructs were grown on SD selective medium for 2 days. Cells were transferred to Whatman No. 1 ¢lter paper, immersed in liquid nitrogen for 10 s to permeabilize cells, and placed on top of another ¢lter which was presoaked in a mixture of 1.8 ml Z-bu¡er containing 5 Wl L-mercaptoethanol and 45 Wl of 20 mg/ml Xgal (5-bromo-4-chloro-3-indolyl-L-D-galactoside) in N,Ndimethylformamide. Plates were sealed with para¢lm and incubated at 30³C. Color development was recorded after a 4-h incubation. 3. Results 3.1. Murine MMS2 and CROC1 cDNAs Screening of the two mouse VYES cDNA libraries V13 and D10 revealed a nearly complete ORF in clone V13-4 that was highly conserved with hMMS2. This clone appeared to lack the ¢rst 15 nucleotides of the ORF based on information from an hMMS2 pseudogene that had been previously isolated (data not shown). After the ¢rst 15 nucleotides were restored to the cDNA clone, the resulting mouse ORF encoded a 145-amino-acid protein that was 97.9% (142/145) identical to hMMS2 and approximately 50% identical to yeast Mms2 (Fig. 1). The same libraries were also screened for sequences homologous to CROC1 using CROC1B cDNA as a probe. Two positive clones, named D10-3 and D10-9, were isolated and their inserts were essentially identical, with D109 being a few nucleotides longer than D10-3 at the 5P end
Fig. 1. Amino acid sequence alignment of S. cerevisiae Mms2 (GenBank accession no. U66724), mMMS2 (accession no. AF303828), hMMS2 (accession no. AF049140), mCROC1 (accession no. AF303829) and hCROC1B (accession no. U39361). The report was generated by a Clustal method with the DNASTAR MegAlign program. It should be noticed that the mCROC1 sequence was assembled from two separate cDNA clones. The C-terminal portion derived from clone D10-9 starts at Val-85 (marked by *).
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of the coding region. Sequence analysis showed that the two clones were highly similar to CROC1B at the nucleotide sequence level. However, they do not appear to contain a full-length mCROC1 coding region, since they lack homology with the N-terminus of CROC1A or CROC1B, and lack a translation start codon. D10-9 contains 137 codons with 90.5% (124/137) sequence identity to C-terminal CROC1 and 84.7% (116/137) identity to mMMS2 (Fig. 1). Rapid ampli¢cation of cDNA ends (RACE) failed to extend further the 5P end of the D10-9 insert. Initial search of the mouse EST database using as a query the CROC1A and CROC1B 5P sequences missing in clone D10-9 revealed two EST clones. One of these clones was isolated by the Washington University^NHMI Mouse EST project from the Stratagene mouse skin cDNA library (#937313). We obtained this EST clone from the ATCC and determined the nucleotide sequence of the entire cDNA insert. The clone could encode 84 amino acids sharing 92.9% (78/ 84) sequence identity with the corresponding CROC1B Nterminus. It does not overlap with D10-9, but instead together they form a continuous sequence to create a complete ORF highly homologous to CROC1B without any nucleotide gaps (Fig. 1). A further search of mouse CROC1 homologs in the EST database using full-length CROC1B as the query showed that out of 40 putative mCROC1 ESTs homologous to the D10-9 insert, 63% (25/40) have 5P ends identical in length to D10-9, 88% (35/40) are within a few nucleotides, whereas the rest are even shorter. Fourteen ESTs were homologous to the above ATCC EST. Of these, seven had the same 3P end, while the other seven were shorter than the ATCC EST. Hence, there is yet no evidence that the 5P and 3P mouse cDNAs homologous to CROC1B are physically linked. 3.2. Tissue distribution of mMMS2 and mCROC1 A mouse multiple tissue Northern membrane was hybridized with CROC1B and hMMS2 to gain insight into the transcript size, the number of transcripts and tissue speci¢city of these UEVs. Although hMMS2 and CROC1 are greater than 90% identical within their core domains, their cDNA probes do not cross-hybridize [9] and therefore the same membrane can be used to compare their transcripts. hMMS2 shares 95% nucleotide sequence identity with mMMS2 in the coding region and hybridized to a major 1.4 kb mouse transcript (Fig. 2A) plus a few minor transcripts of 2.0, 3.4 and 4.0 kb, with an additional 1.0 kb major transcript displayed in the testis. This is in contrast to human tissues where only a 1.5 kb transcript was observed from all tissues examined, including testis ([9], and data not shown). Unlike human CROC1, which has two distinct transcripts, CROC1A (2.0 kb) and CROC1B (2.1 kb) [6,7], two distinct mouse CROC1 transcripts could only be found in kidney (2.4 and 2.0 kb) and testis (2.0 and 1.5 kb). All other tissues displayed only a
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Fig. 2. Expression of mMMS2 and mCROC1 in di¡erent mouse tissues. The membrane containing multiple tissue poly(A) RNA (2 Wg per lane) was sequentially hybridized with (A) the hMMS2 probe, (B) the CROC1B probe, and (C) the L-actin probe. Probe was stripped from the membrane before the next round of hybridization as instructed by the membrane supplier.
single 2.0 kb transcript (Fig. 2B). Both probes showed strong hybridization to mouse heart, brain liver, skeletal muscle, kidney and testis while the spleen expressed extremely low levels of both transcripts (Fig. 2). Overall, the relative tissue distribution and transcript size of mMMS2 and mCROC1 were rather di¡erent from their human homologs. Human CROC1B shares 86% nucleotide sequence identity with the coding region of D10-9 cDNA, and 87% nucleotide sequence identity with that of the putative N-terminal mCROC1 coding region. Hence, the CROC1B probe likely recognizes both mRNA sequences. To further address whether or not both CROC1B homologous sequences exist in a single mouse mRNA molecule, we performed Northern hybridization as described above using N- and C-terminal-speci¢c mouse cDNA probes. To our surprise, the C-terminal cDNA probe (from D10-9) only
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Fig. 3. mMMS2 protects mms2 cells from killing by DNA damaging agents. Yeast strains FY86 (F), FY86mms2 (8) and FY86mms2 transformed with either vector pYES2.0 (a) or pYES-mMMS2 (O) were treated with (A) UV at di¡erent doses or (B) 0.3% MMS for the given times. (C) Yeast strains DBY747 (F, WT), SBL (8, mms2), WXY384 (W, rev3), WXY665 (R, mms2 rev3) and WXY665 transformed with either vector YEp126 (a) or YEpmMMS2 (b) were treated with 0.2% MMS for the given times. Percent survival was scored after a 3-day incubation on YPD plates. The results are the average of at least three independent experiments with standard deviations shown as error bars.
hybridized with the 2.0 kb mRNA in all tissues examined, whereas the N-terminal cDNA probe only recognized the 2.4 kb band in kidney and the 1.5 kb band in testis (data not shown). Hence, this ¢nding further strengthens our previous speculation that the 5P and 3P mouse CROC1B homologous sequences are not physically linked. 3.3. mMMS2 and mCROC1 complement the yeast mms2 null mutant As a ¢rst step in addressing the roles of UEVs in the mouse, we wished to determine if the two cloned mouse MMS2 homologs are able to complement the corresponding yeast mutant. Three di¡erent assays, which had previously been used to characterize the mms2 mutant [5], were carried out. The mms2 null mutant is sensitive to DNA damaging agents such as MMS and UV. The mouse MMS2 ORF was cloned behind an inducible GAL1 promoter to provide high level expression in a Gal yeast mutant strain. Results presented in Fig. 3 demonstrate that mMMS2 fully restored the yeast mms2 mutant to the wild-type level of resistance to killing by UV (Fig. 3A) and MMS (Fig. 3B). However, the yeast mms2 mutant did not display a severe sensitivity to MMS and UV, which is attributed to the functional mutagenesis pathway represented by a nonessential Rev3 DNA polymerase j backing up the loss of the MMS2 error-free PRR pathway [5,10]. Yeast cells carrying both rev3 and mms2 mutations become extremely sensitive to DNA damaging agents, due to simultaneous inactivation of both the error-free and error-prone PRR pathways [5,10]. To demonstrate that mMMS2 functions in the error-free PRR pathway, a mms2 rev3 double mutant was created and used as a recipient to constitutively express mMMS2. Results from MMS killing experiments (Fig. 3C) show that mMMS2 rescued the mms2 rev3 double mutant from killing by
MMS to a level comparable to the rev3 single mutant. These results indicate that (i) mMMS2 indeed functions in the error-free PRR pathway in yeast ; (ii) mMMS2 can functionally replace yeast MMS2; and (iii) high level constitutive expression of mMMS2 does not alleviate defects due to loss of other DNA damage tolerance pathway(s). Finally, one of the most surprising e¡ects of the mms2 mutation is its enhanced spontaneous mutation rate compared with wild-type cells. In this experiment, the mms2 mutant displayed a greater than 50-fold increase in spontaneous Trp reversion rate. Expression of mMMS2 in this strain signi¢cantly reduced the mutation rate (Table 1). A few fold increase above the wild-type level was probably due to loss of the YEp-mMMS2 in a small portion of transformants during nonselective growth. Like mMMS2, mCROC1 isolated from D10-9 was also able to rescue the yeast mms2 mutant from killing by MMS, as shown in Fig. 4. It is interesting to note that mCROC1 used in this experiment is similar in length and sequence to the arti¢cially truncated CROC1B(v80) in a previous study [9].
Table 1 Saccharomyces cerevisiae spontaneous mutation rates Straina
Mutation rate (U1038 )b
Relative ratec
DBY747 (WT) SBL (mms2) SBL/YEp-mMMS2
1.49 þ 1.32 76.5 þ 4.2 6.2 þ 4.0
1 51.3 4.2
a All strains were derived from wild-type DBY747, which carries the revertible trp1-289 amber mutation. b The spontaneous mutation rate is expressed as the number of revertants per cell per generation. The results are an average of three separate experiments with standard deviations. c Relative to the wild-type rate.
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Fig. 4. Protection of mms2 cells by mCROC1 from MMS-induced killing. Yeast strains FY86 (lane 1), FY86mms2 (lane 2), FY86mms2/pYES-mCROC1 (lane 3) and FY86mms2/pYES2.0 (lane 4) were imprinted onto gradient plates containing either YPGal or YPGal+0.03% MMS and the plates were incubated for 42 h at 30³C before photographing. The arrow points towards the higher MMS concentration.
3.4. mMMS2 and mCROC1 interact with Ubc13 It has been reported recently that Ubc13 catalyzes a unique Lys63 polyUb chain assembly and that this activity requires its in vitro physical association with Mms2 [11]. To see if cloned mouse UEVs also function by forming a heterodimer with Ubc13, we performed an in vivo twohybrid analysis (Fig. 5). Yeast cells carrying mMMS2,
mCROC1 or UBC13 alone fused to Gal4DB or Gal4AD coding sequences were unable to transactivate the endogenous PGAL -lacZ reporter gene. In contrast, if a Y190 cell simultaneously carried both Gal4BD -mMMS2 (or Gal4BD mCROC1) and Gal4AD -UBC13, it expressed strong L-gal activity indicative of stable mMMS2 (or mCROC1)-Ubc13 complex formation in vivo. This result shows that both mMMS2 and the truncated mCROC1 are able to interact
Fig. 5. Interaction of mCROC1 and mMMS2 with Ubc13 by a two-hybrid ¢lter assay. S. cerevisiae strain Y190 was cotransformed with pG4BDmCROC1/pGAD-UBC13, pG4BD-mMMS2/pGAD-UBC13, pG4BD-mCROC1/pGAD424, pG4BD-mMMS2/pGAD424, or pG4BD-0/pGAD-UBC13. At least ¢ve independent colonies from each transformation were analyzed and two representative colonies are shown. The positive control was Y190 transformed with pCL2 (YCp-GAL4) and the negative control was Y190 cotransformed with pG4BD-0/pGAD424. The ¢lter was incubated in the Xgal solution at 30³C for 4 h.
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with Ubc13 and probably act as positive regulators of Ubc13 activity. 4. Discussion Genes encoding Ubc variants have been discovered only recently and form at least two families. One family, represented by TSG101, was isolated through screening of mouse tumor suppressor genes [19] and its human homolog has been linked to cancers [20,21]. TSG101 family proteins contain a domain with signi¢cant homology to Ubcs and it has been postulated that they are regulatory proteins in the ubiquitination pathway [22,23]. The second UEV family, represented by Mms2 [5], consists of small proteins in which the entire sequence forms a tertiary structure highly resembling a Ubc core domain (M. Ellison, W. Xiao, unpublished results). Given that the yeast Mms2 is a positive regulator of Ubc13 in a unique polyubiquitination process [11], functions in the error-free PRR pathway and limits spontaneous and damage-induced mutagenesis [5], it is surprising that CROC1, one of its human homologs, was isolated from several laboratories as a putative proto-oncogene [6^8]. To reconcile the aforementioned two seemingly con£icting observations, we propose two alternative hypotheses. The ¢rst hypothesis is that although Ubc13/Mms2 homologs from di¡erent species share signi¢cant homology and perform similar biochemical reactions, their target molecules are di¡erent in human and yeast cells. While the Ubc13-Mms2 targets in yeast cells are PRR proteins, those in human cells may be involved in cellular processes such as cell cycle progression and tumorigenesis. Indeed, it was recently shown that while yeast Ubc13 interacts with a DNA repair RING ¢nger domain protein Rad5 [24], human UBC13 and CROC1/hMMS2 are required for the RING ¢nger protein TRAF6-mediated activation of IUB kinase [13]. The second hypothesis is that CROC1 and hMMS2 may confer di¡erent functions. While CROC1 is a putative proto-oncogene, hMMS2 may be exclusively involved in DNA repair. This hypothesis is consistent with our recent observation that suppression of hMMS2 in human cells by antisense RNA results in phenotypes characteristic of error-free PRR defects (Z. Li, V. Maher, W. Xiao, unpublished results). Although evidence to date favors the above hypotheses, we do not rule out other possibilities. For example, CROC1 and hMMS2 may be multifunctional in a manner reminiscent of the human major DNA repair AP endonuclease, APE/HAP1 [25], also identi¢ed as Ref1, a bifunctional protein capable of stimulating sequencespeci¢c AP-1 DNA binding activity [26]. As an initial step towards understanding the biological and tumorigenic roles of mammalian MMS2 homologs in a whole animal model, we have reported here the molecular cloning of both mCROC1 and mMMS2 cDNAs and characterized their functions in yeast cells. Several issues
raised from this study are discussed below. First of all, we have shown that as in human cells, the mouse has two distinct Mms2 homologs with a very high degree of amino acid identity but signi¢cant nucleotide divergence from each other. This observation indicates the existence of two distinct subfamilies within the MMS2 gene family and that each of the subfamilies may have unique biological functions. It also suggests that the mouse is probably a suitable model for characterizing the MMS2 gene family and its roles in human diseases. Secondly, a few di¡erences have been noticed in the expression of the mouse and human MMS2 homologs, particularly with respect to CROC1. These include lack of alternative CROC1 transcripts in all mouse tissues examined, as well as di¡erences in tissue distribution of both genes in human and mouse. Lastly, probably the most surprising observation is that all mCROC1 cDNAs so far isolated from di¡erent tissues and cell cultures are truncated at the site separating the Mms2homologous core domain and the N-terminal coding sequences. cDNAs encoding both sequences have been identi¢ed that would form a continuous CROC1B homolog; however, there is no evidence that they are physically linked in a single mRNA molecule. Furthermore, mouse mRNAs from di¡erent tissues exclusively hybridize with either N-terminal or C-terminal-speci¢c probes, but not both. The cDNA encoding the core domain mCROC1 lacks a translation start, but is able to complement the corresponding yeast mms2 mutant when supplemented with a translation start codon. A few scenarios may be considered to explain these observations. (i) The mCROC1 5P and 3P coding sequences are in two di¡erent exons that may be joined rarely, ine¤ciently, only in some tissues, or only under particular environmental stress (e.g., DNA damage). (ii) These two cDNAs, although both are homologous to di¡erent regions of CROC1B, are from two distinct mouse genes. This scenario is less likely since the 5P coding cDNA does not have a stop codon and the 3P coding cDNA does not have a start codon; hence, we have to assume further that both cDNAs represent stand-alone exons that have to be further processed or joined to other transcripts. (iii) Given our previous observation that the N-terminally truncated, but not the full length CROC1B, can replace yeast MMS2 functions [9], it is possible that the N-terminal mCROC1 coding sequence is a regulatory element of mCROC1 activity and that the truncated cDNAs correspond to post-transcriptional modi¢cation of the full-length mCROC1. It is further proposed that this post-transcriptional regulation of mCROC1 activity may be replaced in human cells by alternative splicing that yields two distinct CROC1 transcripts. In view of this hypothesis, it is interesting to note that CROC1A is reported to be functional in yeast [27] while CROC1B is not [9]. Recent studies [24,28] have revealed that the yeast PRR and mutagenesis pathway involves at least two Ubc (Ubc2/Rad6 and Ubc13-Mms2) and two RING-¢nger do-
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main proteins (Rad5 and Rad18) and that these proteins interact physically and genetically. So far only one transgenic mouse model (HR6B) has been reported [29]. However, this model has not been informative with respect to PRR, probably due to the second mouse Rad6 homolog HR6A [30]. Molecular cloning of mMMS2 and mCROC1 genomic DNA in conjunction with the mouse genome project may shed light on this issue. Hence, creation of transgenic mice de¢cient in mMMS2 and/or mCROC1 will address how these genes are involved in diverse cellular processes including cell cycle progression, di¡erentiation, immortalization and DNA postreplication repair. Acknowledgements The authors wish to thank Dr. S. Elledge for mouse cDNA libraries and other laboratories for reagents. This work was supported by Medical Research Council of Canada operating grant MT-15076 to W.X. J.F. was supported by a University of Saskatchewan College of Medicine Graduate Fellowship and C.A. was supported by a University of Saskatchewan College of Medicine Postdoctoral Fellowship. References [1] A. Ciechanover, The ubiquitin-proteasome proteolytic pathway, Cell 79 (1994) 13^21. [2] A. Alves-Rodrigues, L. Gregori, M.E. Figueiredo-Pereira, Ubiquitin, cellular inclusions and their role in neurodegeneration, Trends Neurosci. 21 (1998) 516^520. [3] A. Ciechanover, A. Orian, A.L. Schwartz, The ubiquitin-mediated proteolytic pathway : mode of action and clinical implications, J. Cell. Biochem. 34 (Suppl.) (2000) 40^51. [4] E.T. Yeh, L. Gong, T. Kamitani, Ubiquitin-like proteins: new wines in new bottles, Gene 248 (2000) 1^14. [5] S. Broom¢eld, B.L. Chow, W. Xiao, MMS2, encoding a ubiquitinconjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway, Proc. Natl. Acad. Sci. USA 95 (1998) 5678^5683. [6] L. Ma, S. Broom¢eld, C. Lavery, S.L. Lin, W. Xiao, S. Bacchetti, Upregulation of CIR1/CROC1 expression upon cell immortalization and in tumor-derived human cell lines, Oncogene 17 (1998) 1321^1326. [7] M.L. Rothofsky, S.L. Lin, CROC-1 encodes a protein which mediates transcriptional activation of the human FOS promoter, Gene 195 (1997) 141^149. [8] E. Sancho, M.R. Vila, L. Sanchez-Pulido, J.J. Lozano, R. Paciucci, M. Nadal, M. Fox, C. Harvey, B. Bercovich, N. Loukili, A. Ciechanover, S.L. Lin, F. Sanz, X. Estivill, A. Valencia, T.M. Thomson, Role of UEV-1, an inactive variant of the E2 ubiquitin-conjugating enzymes, in in vitro di¡erentiation and cell cycle behavior of HT-29M6 intestinal mucosecretory cells, Mol. Cell. Biol. 18 (1998) 576^589. [9] W. Xiao, S.L. Lin, S. Broom¢eld, B.L. Chow, Y.F. Wei, The products of the yeast MMS2 and two human homologs (hMMS2 and CROC-1) de¢ne a structurally and functionally conserved Ubc-like protein family, Nucleic Acids Res. 26 (1998) 3908^3914. [10] W. Xiao, B.L. Chow, T. Fontanie, L. Ma, S. Bacchetti, T. Hryciw, S. Broom¢eld, Genetic interactions between error-prone and error-free postreplication repair pathways in Saccharomyces cerevisiae, Mutat. Res. 435 (1999) 1^11.
77
[11] R.M. Hofmann, C.M. Pickart, Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair, Cell 96 (1999) 645^653. [12] J. Brusky, Y. Zhu, W. Xiao, UBC13, a DNA-damage-inducible gene, is a member of the error-free postreplication repair pathway in Saccharomyces cerevisiae, Curr. Genet. 37 (2000) 168^174. [13] L. Deng, C. Wang, E. Spencer, L. Yang, A. Braun, J. You, C. Slaughter, C. Pickart, Z.J. Chen, Activation of the IUB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain, Cell 103 (2000) 351^361. [14] S.J. Elledge, J.T. Mulligan, S.W. Ramer, M. Spottswood, R.W. Davis, VYES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations, Proc. Natl. Acad. Sci. USA 88 (1991) 1731^1735. [15] F. Sherman, G.R. Fink, J. Hicks, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1983. [16] J. Hill, K.A. Donald, D.E. Gri¤ths, G. Donald, DMSO-enhanced whole cell yeast transformation, Nucleic Acids Res. 19 (1991) 5791. [17] M.S. Williamson, J.C. Game, S. Fogel, Meiotic gene conversion mutants in Saccharomyces cerevisiae. I. Isolation and characterization of pms1-1 and pms1-2, Genetics 110 (1985) 609^646. [18] P.L. Bartel, S. Fields, Analyzing protein-protein interactions using two-hybrid system, Methods Enzymol. 254 (1995) 241^263. [19] L. Li, S.N. Cohen, Tsg101 : a novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells, Cell 85 (1996) 319^329. [20] L. Li, X. Li, U. Francke, S.N. Cohen, The TSG101 tumor susceptibility gene is located in chromosome 11 band p15 and is mutated in human breast cancer, Cell 88 (1997) 143^154. [21] Y. Oh, M.L. Proctor, Y.H. Fan, L.K. Su, W.K. Hong, K.M. Fong, Y.S. Sekido, A.F. Gazdar, J.D. Minna, L. Mao, TSG101 is not mutated in lung cancer but a shortened transcript is frequently expressed in small cell lung cancer, Oncogene 17 (1998) 1141^1148. [22] C.P. Ponting, Y.D. Cai, P. Bork, The breast cancer gene product TSG101 : a regulator of ubiquitination ?, J. Mol. Med. 75 (1997) 467^469. [23] E.V. Koonin, R.A. Abagyan, TSG101 may be the prototype of a class of dominant negative ubiquitin regulators, Nat. Genet. 16 (1997) 330^331. [24] H.D. Ulrich, S. Jentsch, Two RING ¢nger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair, EMBO J. 19 (2000) 3388^3397. [25] B. Demple, T. Herman, D.S. Chen, Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: de¢nition of a family of DNA repair enzymes, Proc. Natl. Acad. Sci. USA 88 (1991) 11450^11454. [26] S. Xanthoudakis, T. Curran, Identi¢cation and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity, EMBO J. 11 (1992) 653^665. [27] T.M. Thomson, H. Khalid, J.J. Lozano, E. Sancho, J. Arino, Role of UEV-1A, a homologue of the tumor suppressor protein TSG101, in protection from DNA damage, FEBS Lett. 423 (1998) 49^52. [28] W. Xiao, B.L. Chow, S. Broom¢eld, M. Hanna, The Saccharomyces cerevisiae RAD6 group is composed of an error-prone and two errorfree postreplication repair pathways, Genetics 155 (2000) 1633^ 1641. [29] H.P. Roest, J. van Klaveren, J. de Wit, C.G. van Gurp, M.H. Koken, M. Vermey, J.H. van Roijen, J.W. Hoogerbrugge, J.T. Vreeburg, W.M. Baarends, D. Bootsma, J.A. Grootegoed, J.H. Hoeijmakers, Inactivation of the HR6B ubiquitin-conjugating DNA repair enzyme in mice causes male sterility associated with chromatin modi¢cation, Cell 86 (1996) 799^810. [30] M.H. Koken, E.M. Smit, I. Jaspers-Dekker, B.A. Oostra, A. Hagemeijer, D. Bootsma, J.H. Hoeijmakers, Localization of two human homologs, HHR6A and HHR6B, of the yeast DNA repair gene RAD6 to chromosomes Xq24-q25 and 5q23-q31, Genomics 12 (1992) 447^453.
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