Associations of UBE2I with RAD52, UBL1, p53, and RAD51 Proteins in a Yeast Two-Hybrid System

hances their resistance to radiation (Park, 1995). HuThe yeast RAD52-dependent pathway is involved in man RAD52 has at least two independent functional DNA recombination and double-strand break repair. domains that mediate self-association (Shen et al., Yeast ubiquitin-conjugating enzyme UBC9 partici1996b) and interaction with human RAD51 (Shen et pates in Sand M-phase cyclin degradation and mitotic al., 1996a). control. Using the human RAD52 protein as the ‘‘bait’’ Ubiquitin-conjugating enzymes constitute a family in a yeast two-hybrid system, we have identified a huof proteins, including RAD6 (UBC2) and cdc34 (UBC3), man homolog of yeast UBC9, designated UBE2I, that that specifically conjugate ubiquitin to selected prointeracts with RAD52, RAD51, p53, and a ubiquitin-like teins (Hershko, 1991; Jentsch, 1992; Jentsch et al., protein UBL1. These interactions are UBE2I-specific, 1990; Koken et al., 1991). Many crucial biochemical since another DNA repair-related ubiquitin-conjugatprocesses, including selective protein degradation, ing enzyme, RAD6 (UBC2), does not interact with these apoptosis, cell cycle control, ribosome biogenesis, and proteins. The interaction of UBE2I with RAD52 is meDNA repair are regulated by ubiquitination (for review, diated by RAD52’s self-association region. These resee Finley and Chau, 1991; Jentsch, 1992). RAD6 parsults suggest that the RAD52-dependent processes, cell ticipates in DNA repair presumably by ubiquitinating cycle control, p53-mediated pathway(s), and ubiquitihistones and associating with RAD18 proteins (Finley nation interact through human UBE2I. q 1996 Academic and Chau, 1991; Jentsch, 1992). Press, Inc. In an effort to identify a novel protein(s) in this repair pathway, we used a yeast two-hybrid system to screen for proteins that may associate with the human RAD52 INTRODUCTION protein. We identified the human homolog of yeast ubiCellular exposure to DNA damaging agents induces quitin-conjugating enzyme UBC9 through its interacmany cellular processes, including DNA damage retion with the human RAD52 protein. This human gene pair. The RAD52 epistasis gene group is required for has been assigned the symbol UBE2I by the Human DNA double-strand break (DSB) repair and mitotic/ Gene Nomenclature Committee. meiotic recombination. Many yeast proteins that participate in the RAD52-dependent DSB repair pathway MATERIALS AND METHODS have been identified. However, only two human counterparts of these proteins have been reported, i.e., Yeast two-hybrid system. Materials and methods for yeast twoRAD51 and RAD52 (Muris et al., 1994; Shen et al., hybrid systems used in this study have been extensively described in our previous reports (Shen et al., 1996a,b,c); therefore, they will 1995; Shinohara et al., 1993). The human RAD51 probe discussed only briefly here. The yeast strains SFY526 (Clontech tein shows many similarities to yeast RAD51, including Laboratories, Palo Alto, CA) were used to examine the interaction DNA binding activity and interaction with the human of two known proteins. b-galactosidase (LacZ) activity, representing RAD52 protein (Benson et al., 1994; Shen et al., 1996a). the interaction of two fusion proteins in the yeast two-hybrid system, Overexpression of human RAD52 in monkey cells enwas measured by color development in filter assays according to the Matchmaker Kit manual (Clontech Laboratories) and previous reports (Shen et al., 1996a,b,c). Yeast HF7c and a pACT vector-based 1 To whom correspondence should be addressed at LS-6, MS M888, cDNA library were used to screen a cDNA for RAD52-interacting Life Sciences Division, Los Alamos National Laboratory, Los Alamos, proteins using procedures described in the two-hybrid system manNM 87545. Telephone: (505) 667-2789. Fax: (505) 665-3024. E-mail: ual (Clontech Laboratories Inc.) and a previous report (Shen et al., dchen@telomere.lanl.gov. 1996c). 2 Abbreviations used: DSB, DNA double-strand break; UBE2I, a human homolog of the yeast ubiquitin-conjugating enzyme UBC9; Cloning of RAD6 cDNA. The RAD6 two-hybrid construct was cloned by two consecutive PCR amplifications. For the primary PCR UBL1, a 12-kDa ubiquitin-like protein; Gal4-DA, the Gal-4 DNA activation domain (amino acids 768–881); Gal4-DB, the Gal-4 DNA analysis, human RAD6 cDNA was amplified from a cDNA pool isolated from the pACT cDNA library (Clontech) by using primers binding domain (amino acids 1–147); LacZ, b-galactosidase gene.


INTRODUCTION
protein. We identified the human homolog of yeast ubi-Cellular exposure to DNA damaging agents induces quitin-conjugating enzyme UBC9 through its interacmany cellular processes, including DNA damage retion with the human RAD52 protein. This human gene pair. The RAD52 epistasis gene group is required for has been assigned the symbol UBE2I by the Human DNA double-strand break (DSB) 2 repair and mitotic/ Gene Nomenclature Committee. meiotic recombination. Many yeast proteins that participate in the RAD52-dependent DSB repair pathway

MATERIALS AND METHODS
have been identified. However, only two human counterparts of these proteins have been reported, i.e., Yeast two-hybrid system. Materials and methods for yeast two- RAD51 and RAD52 (Muris et al., 1994; hybrid systems used in this study have been extensively described in our previous reports (Shen et al., 1996a,b,c); therefore, they will 1995; Shinohara et al., 1993). The human RAD51 probe discussed only briefly here. The yeast strains SFY526 (Clontech tein shows many similarities to yeast RAD51, including Laboratories, Palo Alto, CA) were used to examine the interaction DNA binding activity and interaction with the human of two known proteins. b-galactosidase (LacZ) activity, representing RAD52 protein (Benson et al., 1994;Shen et al., 1996a). the interaction of two fusion proteins in the yeast two-hybrid system, Overexpression of human RAD52 in monkey cells enwas measured by color development in filter assays according to the Matchmaker Kit manual (Clontech Laboratories) and previous reports (Shen et al., 1996a,b,c). Yeast HF7c and a pACT vector-based 1 To whom correspondence should be addressed at LS-6, MS M888, cDNA library were used to screen a cDNA for RAD52-interacting Life Sciences Division, Los Alamos National Laboratory, Los Alamos, proteins using procedures described in the two-hybrid system man-NM 87545. Telephone: (505) 667-2789. Fax: (505) 665-3024. E-mail: ual (Clontech Laboratories Inc.) and a previous report (Shen et al., dchen@telomere.lanl.gov. 1996c). 2 Abbreviations used: DSB, DNA double-strand break; UBE2I, a human homolog of the yeast ubiquitin-conjugating enzyme UBC9; Cloning of RAD6 cDNA. The RAD6 two-hybrid construct was cloned by two consecutive PCR amplifications. For the primary PCR UBL1, a 12-kDa ubiquitin-like protein; Gal4-DA, the Gal-4 DNA activation domain (amino acids 768-881); Gal4-DB, the Gal-4 DNA analysis, human RAD6 cDNA was amplified from a cDNA pool isolated from the pACT cDNA library (Clontech) by using primers binding domain (amino acids 1-147); LacZ, b-galactosidase gene. 183 0888-7543/96 $18.00 Copyright ᭧ 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

p53, and UBL1 in the Yeast Two-Hybrid System
ATCTGCGACATGTCCACCCCGGC and a SalI-tagged primer 5TGCAGGTCGACGCTGTACCCGGGGTCAAC were used for am-We further characterized the interaction of UBE2I plification. This purified secondary PCR product was cut with BglII with RAD52 and RAD51 using a different two-hybrid and SalI and inserted into two two-hybrid vectors (pGBT9 and system than the one used in the original screening. between human RAD51/RAD52 and human RAD6. Huat codon 49. Because this sequence was found in all four independent man RAD6 (UBC2) protein was compared with UBE2I clones, Glu being a conserved Gly substitution, and because GAG because it is the human protein with the closest amino (Glu) is normally present in another RAD6 cDNA clone (GenBank acid sequence to UBE2I and because it is a DNA repair Accession No. M74525), we concluded that our RAD6 is a normal copy of human RAD6 cDNA.
enzyme. This result therefore suggests that the interac-Other procedures. DNA sequencing, Northern hybridization, and tion between UBE2I and the human RAD51/RAD52 construction of yeast expression vectors have been described (Shen complex is UBE2I-specific. et al., 1995UBE2I-specific. et al., , 1996a and will be briefly discussed in the figure Since the tumor suppresser protein p53 and a newly legends.
identified ubiquitin-like protein (UBL1) are implicated in the RAD51/RAD52 complex (Gibson et al., 1996; et al., 1996;Shen et al., 1996c), we further tested their associations with UBE2I (Fig. 1). Our data cDNA Cloning of UBE2I, a Homolog of the Yeast show that human UBE2I, and not RAD6, associates Ubiquitin-Conjugating Enzyme UBC9 or Hus5 with p53 and UBL1. Truncated p53 protein (amino acids 72-393) lacking the N-terminal transactivation A 1093-bp cDNA clone (see GenBank Accession No. U38785) was isolated from 0.3 1 10 6 independent region was used to eliminate any false positives that could have resulted from the p53 transactivation do-clones via interaction with human RAD52 in a yeast two-hybrid system. The cDNA codes for an 18-kDa pro-main in the two-hybrid system. tein of 158 amino acids (data not shown). The predicted UBE2I Interacts with RAD52 through the Selfamino acid sequence showed 81% similarity (66% iden-Association Domain of the RAD52 Protein tity) with the Schizosaccharomyces pombe mitosis ubiquitin-conjugating enzyme Hus5 (Al-Khodairy et al.,

Sturzbecher
The human RAD52 protein shares homology with yeast RAD52 only in the N-terminus 1/3 region (Shen 1995), 75% similarity (56% identity) with the Saccharomyces cerevisiae ubiquitin-conjugating enzyme UBC9 et al., 1995). To map the RAD52 region that interacts with UBE2I, a series of RAD52 constructs was used. (Seufert et al., 1995), and 59% similarity (42% identity) with the human RAD6 (UBC2) protein (Koken et al., Figure 2 shows that region 65-165 interacts with UBE2I. This region has been assigned as the RAD52 1991). A signature sequence for all ubiquitin-conjugating enzymes (Hershko, 1991;Jentsch, 1992; Jentsch et self-interaction domain (Shen et al., 1996b). The RAD52 self-interaction region is highly conserved in al., 1990) was found in this protein by searching a protein motif database. Based on these analyses, we con-many organisms (Muris et al., 1994;Shen et al., 1995), inferring that this self-interaction is likely to be very clude that the gene codes for an ubiquitin-conjugating enzyme, i.e., the human homolog of S. cerevisiae UBC9 important for RAD52's function. It is possible that the interaction of UBE2I with the RAD52 protein may tar-or S. pombe Hus5. This human gene is named UBE2I according to the recommendation of the Human Gene get RAD52 for degradation or modulate its function by competing with RAD52 self-association. Nomenclature Committee.

FIG. 2.
The UBE2I-interacting region of the human RAD52 protein is mapped to amino acids 65-165. For details of truncated RAD52 constructs, see Shen et al. (1996b). Other symbols are the same as in Fig. 1. bait in a yeast two-hybrid system. In this report, we UBE2I mRNA Is Highly Expressed in Testis and cloned the same cDNA by using RAD52 as the bait. In Localized at Chromosome 16p13.3 addition, we have shown that UBE2I associates with Northern blotting (Fig. 3) shows that the UBE2I gene RAD52, p53, and UBL1 as well as RAD51. Our data is expressed in many tissues, with the highest mRNA also show that UBE2I is specific in its interactions with level in testis. Mouse and human RAD51 (Shinohara these proteins compared with the RAD6 DNA repair et al., 1993) and chicken RAD52 mRNA (Bezzubova et protein. Another significant finding is that the interacal., 1993) levels are also most elevated in testis. In tion between UBE2I and RAD52 is mediated by addition to the 1.4-kb mRNA band in the Northern blot, RAD52's self-interaction domain, which is highly cona minor band of Ç3.4 kb was detected. This implies served among all RAD52 proteins from different spethat a closely related mRNA species may be expressed cies (Shen et al., 1995(Shen et al., , 1996b. in these human tissues. By using the cDNA as a FISH The tumor suppresser protein p53 has been found to probe, UBE2I was mapped to chromosome 16p13.3 be degraded by a ubiquitination pathway (Blumenfeld (data not shown; available upon request).
et Ciechanover et al., 1994), an 18-kDa rabbit ubiquitin-conjugating enzyme (E2-F1) responsible for DISCUSSION p53 degradation has been purified, and 46 amino acids in three E2-F1 peptide fragments have been obtained Recently, Kovalenko et al. (1996) cloned the human by peptide sequencing (Blumenfeld et al., 1994). It is homolog of the yeast UBC9 gene using RAD51 as the worth mentioning that in the most favorable alignment, only 14 of these 46 amino acids in E2-F1 are identical to UBE2I. In the same regions, there are 34 amino acids in the S. pombe Hus5 protein and 26 amino acids in S. cerevisiae UBC9 identical to human UBE2I. UBE2I is probably not the homolog of rabbit E2-F1, considering that the evolutionary relationship of human to rabbit is closer than that of human to yeast. In S. cerevisiae, the UBC9 protein participates in Sand M-phase cyclin degradation (Seufert et al., 1995). In S. pombe, the Hus5 protein is required for normal mitosis control, and Hus5 mutants show reduced radiation resistance (Al-Khodairy et al., 1995). We have observed that the human RAD52 and RAD51 proteins are highly expressed in S and G2/M phases, but relatively poorly expressed in other phases of the cell cycle (Chen et al., 1996). These observations and the coexpression FIG. 3. A multiple-tissue mRNA blot (Clontech Laboratories) of RAD51/RAD52/UBE2I mRNA in testis (Fig. 3) indiwith 2 mg of mRNA/lane that was sequentially hybridized to UBE2I cate that the association of UBE2I with RAD52/RAD51 cDNA probe (top) or b-actin probe (bottom) according to the manufacturer's protocol. Stripping was performed between hybridizations. is likely to be functionally relevant. However, it is not