Self-association of human RAD52 protein

DNA Repair

ELSEVIER

Mutation Research 364 (1996) 81-89

Self-association of human RAD52 protein Zhiyuan Shen a, Scott R. Peterson a, Jarmon C. Comeaux a, Devon Zastrow a, Robert K. Moyzis b, E. Morton Bradbury “c, David J. Chen aq* ’ Life Sciences Division, LS-6. MS M888, Los Alamos National Laboratory, Los Alamos. NM 87545, USA b Center for Human Genome Studies, LASAlamos National Laboratory Los Alamos. NM 87545, USA ’ Department of Biological Chemist’. School of Medicine, University of California. Dartis, CA 95616, USA Received 5 February

1996; revised 25 April 1996; accepted 29 April 1996

Abstract The yeast RAD.52 protein is required for both homologous DNA recombination and repair of DNA double-strand breaks. RAD52 can bind to the yeast RADS 1 protein, which shares a functional similarity with the bacterial RecA protein. The gene encoding the human homolog of the yeast RAD52 protein shares significant N-terminus amino acid homology with the yeast RAD52 protein. Using a yeast two hybrid system and purified GST-RAD52 fusion protein, we demonstrate that the human RAD52 protein self-associates both in vivo and in vitro. The region of RAD52 required for its self-interaction, mapped here as amino acid residues 65-165, has significant homology with the yeast RAD52 (52% identity, and 89% similarity), suggesting the importance of self-association for RAD52’s function. Keywords: RAD52; RADSI;

DSB; DNA double-strand

breaks; Two hybrid system

1. Introduction It is widely

believed

that repair

of DNA

double-

recombination requires a large number of proteins. Some of these proteins may form a higher order repair complex (repairsome) that is directly involved in repairing DNA strand breaks. In addition, some of the proteins involved in repair may also initiate a signal transduction pathway that serves to mediate strand

breaks

(DSB)

and homologous

* Corresponding author. Tel.: (505) 667-2789; 3024: E-mail: [email protected] 0921-8777/96/$15.00 Copyright PII SO921-8777(96)00025-O

DNA

Fax: (505) 665-

cell cycle arrests in response to DNA damage or DNA recombination. In yeast, DSB repair requires at least 11 proteins, including products of the RAD52 epistasis group genes (RAD50-RAD57) [l I, MREl 1 [2,3], XRS-2 [4] and MRE-4 [5]. The RAD52 gene plays a very important role in DNA homologous recombination and DNA DSB repair in yeast [l&l. Two human homologs of the yeast RAD52 group genes have been cloned, i.e., RAD51 [7] and RAD52 [8,9]. The human RAD52 gene has been mapped to the telomeric region of human chromosome 12p [8,9]. The N-terminal amino acid sequence of the human RAD52 protein is highly conserved with both yeast and mouse RAD52 [8,9]. However, the Cterminal amino acid sequence is quite divergent. Although the human RAD52 protein may be in-

0 1996 Elsevier Science B.V. All rights reserved.

82

Z. Shen et al. /Mutation

volved in homologous DNA recombination [IO], little is known about its biochemical properties. In order to elucidate the mechanism of DSB repair and DNA recombination, it is essential to define the relationships among these individual proteins, including protein-protein interactions and protein modifications. Once an interaction between these proteins has been identified, detailed mapping of the interaction domain is also of importance for structural characterization of the protein. We have initiated experiments designed to identify the proteinprotein interactions with the human RADSI and RAD52 proteins. Interactions between yeast RADS 1 and RAD.52 proteins have been identified both in vivo [I 1,121 and in vitro [13]. We have recently confirmed that the human RAD52 protein interacts with the human RADSI protein, and the region of RAD52 involved in RAD51 interaction has been mapped [14]. In this paper. we show that the human RAD52 protein also self-associates both in vitro and in vivo, an interaction that has not been observed for the yeast RAD52 protein [ 131. We have determined the region of the RAD52 protein that is required for self-association using the yeast two-hybrid system, which detects protein-protein interaction in yeast in vivo and confirmed the observations in vitro using glutathione S-transferase (GST) fused RAD52 proteins. Therefore, our data show that the human RAD52 protein has at least two biochemical activities: binding with the human RADSI protein and binding with other RAD52 molecules.

2. Materials and methods

2.1. Two hybrid system yeast strains and culture medium /plates The yeast strain SFY526(MAZ’a ura3-52 his3200 ade2-101 lys2-801 trpl-901 leu2-3,112 canr ga14-542 galBO-538 URA3::GALl-lacZ), which has LacZ reporter gene fused downstream of the Gall promoter and Gal4 UAS, was purchased from ClonTech Laboratories (Palo Alto, CA). SD and YPD media were prepared as described by the two-hybrid system manual (ClonTech Laboratories, Palo Alto, CA).

Research 364 ( 19961 81 -KY

2.2. Plasmid Lsectors used to construct fusion protein expression plusmids Plasmids pGAD424( LEU2 1, pGTB9(TRPll were purchased from ClonTech Laboratories (Palo Alto, CA); pGEX-5x-l was purchased from Pharmacia Biotech (Alameda, CA); plasmid pET28c from Novagen (Madison, WI). Plasmid pTBG2 was constructed as described below. pGAD424 was used to fuse Gal4p DNA activation domain (Gal4-DA) to the N-terminus of either full-sized or truncated RAD52 proteins. Plasmid pGBT9 was used to fuse Gal4p DNA binding domain (Gal4-DB) to the N-terminus for RAD52. pGEX-5x-l used to create the GSTRAD52 domain fusion proteins. Plasmid pET28c was used to subclone RAD52 cDNA under the T7 promoter control so that (His),-tagged RAD52 protein can be in vitro translated and expressed in bacteria. Plasmid pTBG2 was used to fuse Gal4-DB to the C-terminus of RAD52 protein. 2.3. Construction

of pTBG2 uector

Two steps were used to construct the pTBG2 vector. Step I. Plasmid pGBT9 (Fig. I) was cut with Hind111 to release the Gal4-DB, the multiple cloning sites, the TADi,, element and part of the TRPl gene. The remaining vector sequences were ligated to a PCR-generated insert containing the multiple cloning sites, the TADHI elements, and the missing part of the TRPI gene. A Hind111 site, a GCA AAG ATG translation starting site, and a KpnI site were tagged at the 5’-end of this PCR fragment. The 3’-end of this PCR fragment covers the Hind111 site in the in TRPl gene. Positive clones were identified by PCR screening and confirmed by DNA sequence analysis. Therefore, a modified pGBT9 vector (pGBTS1 lacking the Gal4-DB was constructed (Fig. I>. Step 2. Plasmid pGBTS was cut with Sal1 and PstI. The Gal4-DB was PCR-amplified and tagged with Sal1 and NlreI at the N-terminus, and a stop codon and PstI site at the C-terminus. This PCR fragment was cut with Sal1 and PstI, then inserted into pGBTS to obtain pTBG2 (Fig. 11. 2.4. Construction

of protein expression

Ljectors

A series of truncated RAD52 protein expression constructs were created by inserting the RAD52

Z. Sherl et nl./Mutntion

cDNA fragments into pre-digested cloning vectors. The RAD52 cDNA fragments were PCR-amplified from a human RAD52 cDNA clone [8] using Nterminal coding sequence primers containing an inframe BumHI site, and C-terminal antisense primers containing a STOP codon and a Sal1 site (See Table 1 for primers). The insert cloned into the pTBG2 vector did not contain a STOP codon in the C-terminal antisense PCR primer. The resultant PCR products were purified with the Qiagen Quick PCR purification Kit (Qiagen, Chatsworth. CA), and double-digested with BamHI and Sal1 (New England Biolabs, Beverly, MA). The restriction enzymes were deactivated at 55°C for 10 min and the digested PCR fragments were purified again using the Quick PCR purification kit. The size and relative quantity of the purified fragments were analyzed by agarose gel electrophoresis. The above digested inserts were then ligated into BamHI/SulI-digested pGBT9, pGAD424, pGEX-5X- 1, pET28c or pTBG2 using T4

SamHI

Hindlll

Hindlll

SEllI PSt I STOP

FIGBTS

GCAAAG Ami

I Hindlll SamHl Sal I PM

I

STOP

pTBG2

GCAAAG ATG

SamHI &Ill

Pst I STOP

Nhel

Fig. I. Construction of pTBG2 vector (See Section 2: Materials and methods for details).

Research 364 (19961 Xl-89

83

DNA ligase (Stratagene, La Jolla, CA). The resultant ligation reactions were transfected into the E. coli strain DH5a (Life Technologies, Inc. Gaithersburg, MD or HBlOI (Life Technologies, Inc. Gaithersburg, MD). Clones were initially screened by PCR and DNA from positive clones was purified using a mini-prep tip (Qiagen, Chatsworth, CA). Correct inframe fusion and insert sequence were confirmed by DNA sequencing using an ABI automatic DNA sequencer (Applied Biosystems Inc. Foster City. CA).

2.5. 01 r:ic,o assq of protein interaction tww hybrid system

wing yeast

To screen for potential interactions between two RAD52 protein fragments, plasmids for the two fusion constructs (one fused with the Gal4-DB, the other fused with Gal4-DA) were co-transfected into yeast cells SFY 526 using the polyetheleneglycol/lithium acetate (PEG/LiAc) method [ 151. Transformed yeast cells were grown on Trp/Leu~ synthetic agar plates for 3 days to select yeast clones bearing both of the fusion vectors. To measure the expression of B-galactosidase ( LacZ) reporter gene, which correlates with the interaction of two fusion proteins expressed from these two vectors, three independent transformants were then transferred to grided filter paper, and grown overnight on Trp -/Leu agar synthetic plates. These clones were then frozen in liquid nitrogen for 5-10 s, placed on top of another filter which was presoaked in Z-buffer (16.1 g/l Na,HPO, 7H,O; 5.5 g/l NaH,PO, . H,O; 0.75 g/l KCI; 0.246 g/l MgSO, 7H,O; pH 7.01 containing 0.27% (v/v) of 2-mercaptoethanol and 0.334% (g/l) of 5-bromo-4-chloro3-indolyl-B-D-galactoside (X-gal), and incubated at 30°C. For each 150 mm filter, 5 ml of Z-buffer was used; and 1.5 ml of Z-buffer was used for a 100 mm filter. Color development was monitored at 4. 8 and 16 h after the incubation was initiated. Clones that developed visible blue color by 4 h were registered as ( + I; 8 h as ( + - 1. If no color developed after I6 h, clones were registered as (-1. Quantitative LucZ activity in Miller’s unit [ 161 was assayed according to the manufacture’s manual of the MatchMaker kit (ClonTech Laboratories, Palo Alto, CA). Briefly, yeast from a single clone were grown in synthetic

84

Z. Shen et al./Mutation

media lacking of Trp/Leu overnight. The density of yeast was determined by measuring the absorbance at 600 nm. Then, 0.1 ml of culture was mixed with 0.7 of Z-buffer, 50 p,l CHCl,, and 50 pl of 0.1% SDS, vortex. 0-Nitrophenylgalactoside (ONPG, 4 mg/ml) was used as substrate for LacZ. After 2 h of incubation at 30°C the reaction mix was centrifuged, and the absorbance of supematant was read at 420 nm. LacZ unit in Miller’s unit was calculated as 1000 X [ A,,,/(t X VX A,,, 1, where t is time of incubation; V is volume of yeast culture; and A,,, is the absorbance of yeast culture at 600 nm. 2.6. Expression

and purification

of GST-fusing

pro-

tein

To express RAD52 GST-fusion proteins, RAD52 cDNAs were inserted into the pGEX-5x-1 vector. A 500 ml HBlOl bacterial culture of each fusion protein construct was grown at 37°C in LB medium containing 100 pg/ml of ampicillin. When the cell culture achieved an optical density at 600 nm of N 0.5, protein expression was induced by the addition of Isopropyl B-D-thiogalactopyranoside (IPTG)

Research 364 (1996181-89

to a final concentration of 0.5 mM and incubated for additional 4 h. The GST-RAD52 fusion proteins were purified using glutathione-conjugated agarose beads (Sigma Biochemical Company, St Louis, MI) as described previously [ 171. Protein concentrations were determined by Bradford analysis and the purified GST fusion protein were stored at - 70°C until further use. 2.7. In vitro protein binding assay Twenty Fg of GST or GST-RAD52 fusion protein were incubated with 100 l.~l of a 1 : 1 slurry of glutathione agarose beads (Sigma Chemical Co., St Louis, MI) in wash buffer (50 mM Tris-HCl, pH 7.9; 200 mM NaCl; 1 mM EDTA, 0.1% Nonidet P-40, 1 mM dithiothreitol) for 1 h at 4°C. The immobilized protein beads were then washed once with 1 ml of wash buffer then stored on ice until further use. Radiolabeled RAD52 protein was produced from the pET28c/RAD52 plasmid using a coupled T7 transcription/rabbit reticulocyte lysate translation system (Promega, Madison, WI> in the presence of 45 pCi of [“Slmethione (New England Nuclear). In

Table 1 Primers used in making RAD52 constructs Position (amino acid)

Primer sequence

Upstream sense primers with BamHI tag

I

CTA TGG ATC CAT AAG ATG TCT GGG ACT GAG

65

CTA TGG ATC CAT TAC ATT GAG GGT CAT CGG

85

CTA TGG ATC CAT GCA CAC TCC ATC ACG CAG

105

CTA TGG ATC CAT GTG GGA GTC TGT GCA TTT

125

CTA TGG ATC CAT GGT TAT GGT GTT AGT GAG

166

CTA TGG ATC CAT ATT CTG GAC AAA GAC TAC

222

CTA TGG ATC CAT GTG ACC TCC CCT TCC AGA

287

CTA TGG ATC CAT AGT GAG GCA GCG CCT CCG

Downstream 418

antisense primers with Sal1 tag TCT GCA GGT CGA CGT CAG TTA AGA TGG ATC AT

301

CTC TGC AGG TCG ACG AGG AGT GCT GTG CGT CAC

301

CTC TGC AGG TCG ACG TTA AGG AGT GCT GTG CGT CAC

221

CTC TGC AGG TCG ACG TTA CTG CTG CAG CTG TGG GTG

165

CTC TGC AGG TCG ACG TTA ACA GTT TCC AAG TGC ATT

1.59

CTC TGC AGG TCG ACG 7TA CCC AAA ACT CCT GAG GGC

132

CTC TGC AGG TCG ACG TTA GAG GCC CTC ACT AAC ACC

124

CTC TGC AGG TCG ACG TTA AAC ATC TTC ATG ATA TGA

Underline sequences denote for the BumHI or Sal1 sites. Italicized sequences represent a STOP codon. Sequences in Bold are from RAD52 cDNA.

Z. Sherz et al. /Mutation

Research 364

Gal4

DNA Activation Domain BAD52 Fusion Rotelns

Gal4

DNA

Fusion G614-DB

Gal4-DAIRAD52

~~~~~~~.:~~~~~~~~~~~~~~~l 52D(, _4, 8)

m m

a

m

B

m

52E(fj5-221) -52Q(125_301)

=52F(287-416)

(27.9*12.4)

+

(292.9*47.2;*“)

-

(27.6f6.4)

+

(527.3+19.1:‘*‘)

+

(160.4f29.5;*“)

_

+

(74.9*12.4;**)

-

+

(99.9f.33.6;‘)

-

_

(34.1i6.3)

_

(14.7to.E)

_

(9.9f6.1)

-

m

03 Gal4

DNA BindIng Domaln

BAD62 Gal4-DE/

Fudon

Proteins

Gal4 DNA Activation Fusbn Proteins GuI4.DA

RAD52

mm

_

-152A(l-124) m

52B(+ttj5)

. .. .. ~-::.Y.z-::.Y.-:.~

52c(t -221)

..... ................................ ......... -

52#-(,_*2,)

520(65-l 65)

Gal4-DB/ RAD52

-

_

52q,_,65) . ... .. .

Ellndlng Domain

Protehts

_

wJ5*/$(,_,q mxj

85

81-89

60 min on ice. The binding reactions were then washed 3 times with 1.0 ml of ice-cold wash buffer, boiled in SDS-PAGE sample buffer and resolved by 10% SDS-PAGE. Radiolabeled RAD52 proteins as-

vitro protein binding reactions were performed by incubating 25 pl (5 kg) of the immobilized GST or GST-fusion proteins with 10 ~1 of the pET28c/RAD52 programmed reticulocyte lysate for

(a)

(1996)

_

Domain

Gel4-DAIRAD52

.. .............................................................. -

(6.2fl.6)

_

(11.3f11.5)

+

(72.2+6.6;***)

+

(231.9f75.9:“‘)

+

(59.4fs.l;*“)

-

+

(79.6f26.1:“‘)

25-30, )

-

_

(79.6t26.1;‘**)

52F(287_418)

-

-

(1 O.st4.3)

-

_

(Not

q (&132)

_

_

m

_

w

520(65165)

w

52E(&_22,) -1

QQ(, m By;:=,*)

(fjfj-159)

dhct6ble)

+

Fig. 2. Interactions between Gal4-DA and GaWDB fusion proteins of RAD52 in yeast two hybrid system using filter assay (see Section 2: Materials and methods for detail). The Gal4 domains were fused at the N-terminus of RAD52 proteins using pGAD424 or pGBT9 vectors. (a) Interaction of full-length RAD52 fused with Gal4-DB to truncated RAD52 fused with GaWDA. (b) Interaction of full-length RAD52 fused with Gal4-DA to truncated RAD52 fused with Gal4-DB. LacZ enzyme activities (Miller Unit “) resulting from interaction between Gal4-DA or GaWDB fusion RAD52 proteins are shown in parenthesis. Average of at least 3 independent colonies + Standard Error are presented. (* * * ) Indicates PI < 0.005; ( * * ) indicates PI < 0.01; and ( * ) for Pl < 0.05. See text for definition of units and definition of PI.

86

Z. Shen et d/Mutation

sociated with the GST-fusion proteins were visualized by autoradiography of the dried gel.

3. Results and discussion In the yeast two hybrid system, interaction between Gal4-DA fused protein with Gal4-DB fused protein will activate LacZ expression. To determine whether the human RAD52 protein has the capacity to bind to other human RAD52 molecules, we cloned the full-length coding region from the RAD52 cDNA into both the Gal4-DB (pGBT9) and the Gal4-DA vectors (pGAD424). In addition to human RAD52 protein, we included human RAD51 protein in these assays. Interactions between these protein combinations measured as LacZ enzyme activities in filter assay are listed in Table 2. Similar to yeast RAD51 protein [12], we found that the human RADSI protein can self-associate in this assay. Human RAD52 protein was also found to self-associate. The specific interaction between human RAD5 1 and RAD52 proteins has been reported [14], and will not be discussed in this report. In order to map the domain of the RAD52 protein involved in self-interaction, a series of truncated Gal4-DA/RAD52 (Fig, 2a) or Gal4-DB/RAD52 (Fig. 2b) fusion constructs were co-transfected with the full-length RAD52 fusion constructs, i.e., Gal4DB/RAD52 (Fig. 2a) or Gal4-DA/RAD52 (Fig. 2b), into the yeast SFY526 strain. Interactions between these fusion proteins measured by LacZ filter

Table 2 Interactions

of RAD52 and RAD5 1 proteins

Gal4-DB Gal4-DB/RADS Ga14-DB/RAD52

1a h

Gal4-DA

Gal4-DA/ RAD51 a

Gal4-DA/ RAD52 h

_c

_

-

+’ +

_ +--c +

a Full-size RADSI protein fused to Gal4 - DA(Gal4DA/RADS 1). or Gal4-DB (GalPDB/RADS 1). b Full-size RAD52 protein fused to Gal4-DA (Cial4-DA/RAD52). or Gal4-DB(GaWDB/RAD52). ’ Interactions presented as LacZ enzyme activity (blue color on filter assay): -. no visible blue color after 16 h of incubation: + -. visible blue color found by 8 h of incubation; +, visible blue color found by 4 h of incubation.

Research 364 (14%) 81-N

assay are shown in Fig. 2. Numbers in the parentheses of the Gal4-DB/RAD52 fusion (Fig. 2a), or Gal4-DA/RAD52 fusion (Fig. 2b) denote the amino acids contained in the constructs. By comparing the results of construct 52A (amino acids I- 124) with 52B (amino acids l-165). we concluded that the region 125- 165 is required for the self-interaction. However, this region does not appear to be sufficient for binding because the construct 52Q(125-301) failed to bind to the full size RAD52 protein. Regions spanned by constructs 520(65-165) and 52E(65-221) bound to the full-length RAD52 protein both in Gal4-DB and Gal4-DA fusion constructs. To confirm the LacZ activity visualized using the filter assay, a quantitative LacZ activity was also employed (see Section 2: Materials and methods for detail). The LacZ activities in Miller’s unit are also shown in Fig. 2. A r-test was performed to test if the LacZ activity resulting from interaction of truncated RAD52 to full-length RAD52 is significantly higher than that of the control ( LucZ activity resulting from interactions of Gal4 domains alone to full-sized RAD52 proteins). Pl is the probability of not significantly higher than the control. Although these quantitative assay correlate with the filter assay very well, it is not intended to show the affinity of these protein interactions. Therefore, in further experiments, only the filter assay will be used. Based on the above data, we propose that the self-interaction domain resides within the region of amino acids 65- 165. To define the C-terminus of RAD52 self-association domain, two more Gal4-DB/RAD52 fusion constructs were made, and their interaction to full length RAD52 is also shown in Fig. 2b. It is inferred from these extra constructs that amino acids 132- 159 are required for the self-association (Fig. 2b). Although only one region was identified as essential for RAD52 self-interaction from the experiments listed in Fig. 2, it is possible that a second region in the full length RAD52 interacts with the domain contained within amino acids 65- 165. As the experiments in Fig. 2 only tested full-size RAD52 with the truncated RAD52 constructs, we performed a second set of experiments to determine whether this domain alone is sufficient to drive the self-interaction when expressed on both fusion protein constructs. The results of this experiment show that region (65-165)

Z. Shen et al. /Mutation

Table 3 Interaction

between two RAD52 fragments

Gal4-DB Fusion

Vecotr 52A( 1 - 124) 52B(l - 165) 52E3(65 - 221) 520(65 - 165) 52U(125 - 165)

(1996)

87

81-89

by filter assay with two hybrid system

Gal4-DA Fusion Vector 52A (I-124) _ _

Research 364

-

52B (l-165)

52E (65-22 1)

520 (65-165)

52U (125-165)

_

_ _

_

+ + + _

+ + + -

+ + + _

_ _ _ -

Symbols as the same in Table 2.

(a)

205kDa =

lgkg

66 IiDa-

45kDa 3lkDa-

(W

Fig. 3. In vitro binding assay of RAD52 proteins. (3) Coomassie blue stain of purified GST-RAD52 fusion proteins. Ten pl of glutathione agarose beads containing 2 pg of RAD52 fusion proteins were denatured and resolved using a 12% SDS-PAGE gel. Numbers in the parenthesis denote amino acids of truncated RAD52 protein. Protein molecular mass marker positions are indicated in kDa (left side). (b) Retained [“SIRAD protein through its interaction with GST/RAD52 fusion proteins bound to glutathione agarose beads. ‘sS-labeled RAD52 protein was produced from the pET28c/RAD52 plasmid using a rabbit reticulocyte lysate T7 RNA polymerase coupled transcription/translation system. Labeled RAD52 protein was incubated with of 5 (Ig GST fusion proteins bound to 25 p,1 glutathione agarose beads, and washed as described in Section 2: Materials and methods. Bound [35S]RAD52 proteins were resolved by SDS-PAGE and visualized by autoradiography of the dried gel. Arrow indicates the position of the labeled RAD52 protein. Lysate denotes 1.0 ~1 of the programmed lysate containing the in vitro translated and labeled RAD52 protein as a standard; Lanes GST, 52B, 52C, 52D, 52E, 52F, 520, 52P, 52R and 52U denote [35S]RAD52 proteins bound to GST protein or the corresponding GST-RAD52 fusion proteins. The region of these GST-fusion protein used for this assay are the same as in panel (a). Protein molecular mass marker positions are indicated in kDa (left side).

88

Z. Sherz et al. /Mutation

is able to interact with itself (Table 3), which suggests that the same region (65 165) involved in the self-interaction. To further define the mapping of N-terminus of the self-interaction domain. Gal4-DB was fused to the C-terminal end of several RAD52 constructs using pTBG2 vector. The ability of these constructs to interact with the full size RAD52/Gal4-DA fusion protein was tested (Table 4). These results show that neither region 125-301 nor 105-301 is able to interact with RAD52. However, regions 85-301 and I-301 are sufficient to interact with RAD52, indicating that region 65-85 is not required for the interaction. To test whether the interaction domain identified by the yeast two-hybrid system represented the direct association of RAD52 monomers, we tested the ability of RAD52 proteins to self-associate in vitro. To do this, we incubated glutathione-agarose immobilized GST-RAD52 fusion proteins with an [ 3”S]methionine labeled full-length RAD52 protein. In this assay, 3”S-labeled RAD52 protein bound to GST-fusion protein should be retained with glutathione agarose beads. Fig. 3 shows the results of experiments testing the association of a variety of GST-RAD52 protein constructs with the labeled RAD52 protein. Fig. 3a illustrates the RAD52 GSTfusion proteins that were bound to glutathione agarose beads. Fig. 3b shows the labeled RAD52 protein retained with different GST-fusion proteins. In agreement with the two hybrid data, we found that GST-RAD52 fusion proteins not containing amino acids 65- 165 (i.e., GST-52F(287-4 181, GST52p(I66-301). GST-52R(222-3011, GST-52U(125165) and GST alone) failed to bind to the full-length RAD52 protein whereas the GST-RAD52 fusion proteins that contain amino acids 65- 165 (i.e., GST-

Table 4 Interactions between full-length RAD52 fused to the N-terminus of Gal4-DB

and RAD52

Gal4-DAGal4-DA/RAD52( Gal4-DB RAD52(125 - 301)/Gal4-DB RAD52(105-301)/Ga14-DB RAD52(85-301)/Gal4-DE RAD52(1- 301)/Gal4 - DB

_

_

-

_ + +

fragments

Research 364 (IYY6) 81-N

52B(l-1651, GST-52C(l-221), GST-52D(l-418, full length), GST-52E(65-2211, and GST-52(X65165)) fusions retained the labeled RAD52 protein. GST alone was used as a negative control. However, in contrast to the two-hybrid data, we found that the ability of the 52B, 52E and 520 domains to bind to the full-length RAD52 protein was compromised in the GST fusion constructs, which may be due to alterations in the conformation of the interaction domain in this fusion context. In light of all presented results, we conclude that RAD52 protein can self-associate and that the protein domain required for this association resides within amino acids 65- 165, and most likely 85- 159. Despite the apparent conservation (52% identity and 89% similarity) of the human RAD52 self-interacting region with the yeast RAD52 protein, yeast RAD52 did not self-associate as witnessed in a previously published report 1131 because yeast RAD52 protein was not retained by a RAD52 column. Thus, this self-association of human RAD52 protein may be an unique property of human RAD52. Conversely, this suggests that the yeast RAD52 may self-associate under some conditions not yet explored by the previous report [ 131. In a previous report, we have found that the RADS l-interacting region of RAD52 protein resides in the C-terminus (amino acids 287-333) [14]. In this report, we have found that self-association of RAD52 proteins can be formed in the absence of the RADS l-interacting region (287-333). Therefore, we conclude that human RAD52 has at least two independent functional domains, i.e.. amino acids 65-165 for self-association, and amino acids 287-333 for interaction with RAD5 1. The biological significance of RAD52 selfassociation is not clear. However, yeast RAD52 is required for almost all homologous recombination, and the self-association domain of RAD52 protein resides in the N-terminus conserved region between yeast and human. By inference, this self-association may be functionally important for RAD52 protein.

I-41 8)

Acknowledgements We wish to thank Ms. Sue Thompson for her assistance in synthesizing primers for PCR and DNA sequencing. and Dr. M.S. Park for helpful discus-

Z. Shen et al./Mutation

sion. ZS was supported by a Director Funded Postdoctoral Fellowship at the Los Alamos National Laboratory. This research was supported by NIH Grant CA50519 to D.J.C.

References [II Friedberg.

E., Siede, W. and Cooper, A. (19911 Cellular respoonse to DNA damage in yeast, in: J. Broach, J.R. Pringle and E. Jones (Eds.1, The Molecular and Cellular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. pp. 147-192. 121Ajimura. M.. Leem, S.H. and Ogawa, H. (19931 Identification of new genes required for meiotic recombination in Saccharomyes cere~~isiae. Genetics, 133. 51-66. 131 Johzuka. K. and Ogawa. H. (1995) Interaction of Mrel 1 and Rad50: two proteins required for DNA repair and meiosisspecific double-strand break formation in Saccharom~ces cerwisioe. Genetics, 139. 1521-1532. 141Ivanov. E.L., Korolev. V.G. and Fabre, F. (1992) XRS?. a DNA repair gene of Saccharomyes cerel,isiae. is needed for meiotic recombination. Genetics. 132, 65 l-64. [51 Leem. S.H. and Ogawa, H. (1992) The MRE4 gene encodes a novel protein kinase homologue required for meiotic recombination in Succhcrromyces crrer~isiae. Nucleic Acids Res.. 20, 349-457. 161Game. J.C. (1993) DNA double-strand breaks and the RAD50-RAD57 genes in Saccharomyces. Semin. Cancer Biol., 4, 73-83. 171 Shinohara. A., Ogawa, H., Matsuda, Y.. Ushio, N.. Ikeo, K. and Ogawa, T. ( 19931 Cloning of human, mouse and fission yeast recombination genes homologous to RADS I and recA. Nature Genet., 4. 239-243. [81 Shen. Z.. Denison, K., Lobb, R., Gatewood, J.M. and Chen.

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Research 364 (1996) 81-89

D.J. (19951 The human

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