A species-specific interaction of Rad51 and Rad52 proteins in eukaryotes

Adv.

A SPECIES-SPECIFIC Rad52

PROTEINS

TOMOKO TOMOATSU Department Toyonaka,

INTERACTION IN

Vol.

OF

31, pp. 93-100

(1995)

Rad51 AND

EUKARYOTES

OGAWA, IKEYA of Biology, Osaka

Biophys.,

AKIRA Faculty

SHINOHARA, of Science, Osaka

AND University,

560, Japan

Genetic recombination is involved in a number of biological processes including maintenance and diversification of the genetic material of living cells. The red gene of Escherichia coli has been studied extensively. It is involved in recombination, repair of DNA damage, and induction of SOS genes (1-5). Since recombination and repair of DNA damage are common and necessary reactions for all living cells, eukaryotic cells are likely to have processes similar to those catalyzed by the RecA protein. In our search for mutants that show similar pleiotropic phenotypes to those of recA mutant affecting recombination and repair of DNA damage among previously isolated rad mutants of Saccharomyces cerevisiae (6), we found rad51 and rad52 mutants belonging to the RAD52 epistasis group. We, therefore, cloned the corresponding wildtype genes and analyzed the functions of the products of these genes (7-9). We also found that the &ID51 homologues are widely distributed in different organisms (10). In this review, we summarize the structures and properties of the Rad51 and Rad52 proteins in eukaryotes and describe the speciesspecific interaction of the proteins.

93

94 I.

T. OGAWA

PROPERTIES

OF RAD.51

AND

RAD52

GENES

ET AL.

OF S. CEREVISIAE

The yeast rad51 or rad52 mutants are defective in both spontaneous and induced mitotic recombination and in meiotic recombination. They are sensitive to treatment with methyl-methane sulfonate (MMS) or ionizing radiation. Synthesis of the Rad51 protein of wildtype cells can be induced by treatment with MMS, while that of the Rad52 protein cannot. With respect to amino acid sequences, the Rad51 protein is homologous to the RecA protein while the Rad52 protein is not, and no similarity of the Rad52 protein to other known proteins was found. Both Tad.51 and rad.52 mutants accumulate 3 times more double-stranded breaks (DSBs) than wild-type cells in meiotic cells, and the efficiency of formation of physical recombinants in these mutants was one-fifth that found in wild-type cells. Thus, the mutants of these genes showed pleiotropic phenotypes similar to those of a recA gene and their functions are required for the repair of DSBs as was also found for the recA gene (2, 11).

II.

THE

PROPERTIES

OF PURIFIED

Rad51

AND

Rad52

PROTEINS

The Rad51 protein binds both single-stranded and double-stranded DNAs in the presence of ATP and it has single-stranded DNA dependent ATPase activity (9). It forms a right-handed nucleoprotein filament on DNA in the presence of ATP. In the filaments, the B-form DNA is extended 1.5 times and unwound (12). For the RecA protein of E. co& the helical nucleoprotein filament is a basic structure that is required for homology search, pairing of homologous sequences and stimulation of repressor cleavage activities of the protein (13, 14). Because of its structural similarity to the RecA protein, the Rad.51 protein is expected to carry out similar reactions. In fact, a strandtransfer activity was recently found for the Rad5 1 protein, although the activity is lower than that of the RecA protein of E. coli and the reaction required for a single-stranded DNA binding protein, RPA-1, of S. cemisiae (15). The Rad52 protein can bind to both single-stranded and doublestranded DNAs in the absence of ATP and carries out annealing of homologous single-stranded DNA. It can also promote the strand transfer reaction. However, the reaction was ATP independent and had an efficiency of the reaction only one-20th that of the RecA protein

(9). We found

that the Rad.51 protein

binds to the Rad52 protein

(7).

SPECIES-SPECIFICITY

OF RADjl

AND

IUD.52

95

FUNCTIONS

Their interaction in an in viva system was also suggested using the two hybrid system (16). These results suggest that the Rad.51 and Rad52 proteins may carry out the reactions of recombination and repair of DNA damage by forming a complex, while the RecA protein can perform similar reactions by itself.

III.

CLONING

OF

MAMMALIAN

MD51

HOMOLOGUES

In an attempt to isolate mammalian Rad51 homologues, we first isolated a Schizosaccharomyces pombe RAD51 homologue (Sprad51) and made the disruption mutant by inserting the wad+ gene of S. pombe to the SpRAD51 gene (Sprad51 ::ura4+). The disruption mutant prevents DNA repair, mitotic recombination, and spore formation as was seen in the S. cerevisiae rad51 mutants (9, 10). Using our knowledge of the homology between S. cerevisiae and S. pombe Rad51 proteins, we cloned human (IIsRadSl), mouse (MmMDSl), and chicken (ChRAD51) IUD51 genes. All IUD51 homologues encode a protein structurally homologous to yeast Rad51 proteins (9, 17). Then, the expression of MD51 mRNA in different tissues of the mouse was examined. High expression of IUD51 in testis and ovary suggests that this gene may be involved in meiotic recombination as it is in yeast. High expression observed in thymus and spleen suggests that the gene could be involved in lymphoid cellspecific reactions, such as recombination during antibody formation. The high expression of RNA in cells during embryogenesis suggests that the gene is involved in DNA metabolism and repair of DNA damage ( 10).

I\~.

COMPARISON

OF THE

PRIMARY

STRUCTURES

OF

Rx%51 HOMOLOGUES

The extent of homology among two mammalian, one chicken, and two yeast RadSl homologues was compared with Dmcl homologues and E. coli RecA protein (Fig. 1). S. cerevisiae Dmcl (Isc2) protein (18, 19) is involved in meiotic recombination, synaptonemal complex formation, and cell cycle progression, but not in mitotic recombination (18). It is homologous to both RadSl of S. cerevisiae and E. coli RecA proteins. LIMZ5 gene of the lily has also been cloned from a cDNA library which was made from mRNA induced in the meiotic cells of the lily, and was identified as a homologue of the DMCl gene (19). All the eukaryotic proteins described here have extended N-terminal regions that are far longer than those of various prokaryotic

96

T.

j Domain

Ii

Domain

L HsRad51, MmRad51 & ChRAD51

OGAWA

ET

AL.

j clwminsl : Reglm

II

3;7 339

NH

coon 5

SpRad51

COOH

0

ScRadll

coon

NH,

RecA

Limld A.TYP

Fig. 1. A diagrammatic in the Rad51 homologues, are described in the text.

representation RecA, Dmcl

BTYF-

of regions of amino (Isc~), and Liml5

acid homology proteins. The

found details

RecA proteins, but they do not have the extended C-terminal region that prokaryotic RecA proteins have. The N-terminal amino acid sequences of RadSl homologues, shown by white boxes in Fig. 1, are characteristic to each species. The amino acid sequences represented by lateral stripes for Rad51 homologues and vertical stripes for Dmcl homologues are called “Domain I”. Domain I and the short C-terminal regions are characteristic of Rad51 or Dmcl homologues. “Domain II” is a region that is homologous among all the proteins in Fig. 1 and is shown by diagonal stripes. This region contains the ATP binding regions shown by black boxes. As described above, the RecA protein of E. coli and the Rad51 proteins of S. cerevisiae are able to form a similar nucleoprotein filament and can carry out a strand-transfer reaction. Because these proteins are homologous only in Domain II, this domain is probably responsible for formation of the helical nucleoprotein filament and ATP-dependent strand transfer activity. Other proteins that have homologous Domain II may form a nucleoprotein filament which may be responsible for reactions such as searching and pairing of homologous sequences.

OF RAD52

SPECIES-SPECIFICITY

V.

COMPARISON

OF

THE

MD52

AND

PRIMARY

97

FUNCTIONS

STRUCTURES

OF

Rad52

HOMOLOGUES

Recently, the RAD52 genes of S. pombe (ZO), chicken (ZI), mouse, and human (Pastink and Buerstedde, personal communications) were cloned. Their primary structures are shown in Fig. 2. The N-terminal half of Rad52 proteins shown by diagonal stripes is highly conserved. The homologies of identical amino acids are 69 % between two yeast proteins, 95 % among mouse, human, and chicken, and 60 % between yeast and vertebrate, while the amino acid sequences of the C-terminal half represented by white bars are characteristic to each species. Their homologies are 6% between yeast and 36.5% between human and chicken. No homology was found between yeast and vertebrate proteins.

VI.

SPECIES-SPECIFICITY

OF

Rad51

PROTEIN

IN

REPAIR

OF

DNA

DAMAGE

The S. pombe and human RAD52 genes are unable to complement the sensitivity to UV light irradiation or to MMS treatment of S. cerevisiae r&51 deletion mutant cells (9, 10). Similarly, the cloned human (HSRADSI) R/ID51 gene that is expressed under the control of a& mutant resistant to promoter of S. pombe cannot make the disruption gene which is also treatment with MMS. However, the S&AD52

ScRad52

SpRad52

HsRad52

ChRad52 Fig. 2. A diagrammatic in the Rad52 homologues.

representation S. ewe&&,

of regions S. pombe,

of amino acid homology human, and chicken Rad52

found pro-

teins are referred Diagonal stripes stripes represent

to as ScRadS2, SpRad52, HsRad52, and ChRad52, respectively. represent homologous regions for all Rad52 proteins. Vertical the homologous region between mouse and chicken. Numbers in

the bar represent bers in parentheses

homology represent

of identical amino the homologies

acids (see details in the text). Numof vertebrate to yeast homologues.

98

T.

1

=a 8 0 .z e u: 3 e .$ I

OGAWA

ET

AL.

7

0:

.l

SpmdSl

::~a4

(psdhscRdD51)

c spad51::um4(padh-*rmis1)

\

.Ol

1

spmd51:%?4

(padh-nsuadsl)

Ii b spradsr

mar

(vector)

.OOl 2

4

Concentration Fig.

3.

6

of MMS (x 10%)

Complementation

of the

ScRAD51, H&AD51

and adh-HsRAD51 genes were constructed.

under

adh promoter

the

of Sprad51::ura4+

deficiency

mutants

genes. Plasmids carrying adh-ScRAD51 The transcription of RAD51 gene

of S. pombe.

A Sprad51

::ura4+

mutant

was

with

adh-

and adhis controlled transformed

with a plasmid carrying each of these genes, and the MMS sensitivity of these transformants was examined by plating of these stationary phase cells on SD-leuplates with or without various concentrations of MMS. Open circles or the black triangle

represent

the

survival

fractions

plasmid containing the adh-HsRAD51 Open squares or black squares shows ing

the

adh-ScRAD.51

pombe wild-type

or adh-Sprad51

Sprad51

of the gene

Sprad51: gene,

or

::ura4+

mutant

carrying

a

the vector plasmid, respectively. :ura4+ carrying a plasmid contain-

respectively.

Black

circles

shows

S.

cells.

expressed by the adh promoter is able to endow the resistance to the mutant (Fig. 3). These genes are transcribed normally in the Sprad51 disruption mutant (data not shown). The failure to complement the deficiency of Scrad51 deletion mutant by SpRAD51 or HsRAD.51 gene and that of Sprud52 disruption mutant by H&AD52 gene shows that the action of IUD51 gene is species-specific. Examination of the suppression of the MMS sensitivity of rad511 missense mutant of S. cerevisiue with the N-terminal deletion mutants of the ScRAD51 gene showed that the mutant of 28 amino acid deletion could partially suppress the sensitivity, while the mutant of 107 amino acid deletion could not. The former mutant could not complement the MMS sensitivity of the Scrad52 deletion mutant.

SPECIES-SPECIFICITY

OF JUD.51

AND

RAD52

FUNCTIOLIS

99

Therefore, a region between 28th and 107th amino acid residues in the Rad51 protein is probably responsible for the suppression. As shown above, the SpRAD51 gene which encodes 35 amino acids shorter than the wild-type ScRad51 protein cannot complement the MMS sensitivity of had51 deletion mutant, while the S&AD52 gene can complement the sensitivity of Sprad51 deletion mutant. On the other hand, the HsRAD51 gene which is 74 amino acids shorter than ScRad51 protein does not complement the MMS sensitivity in either the Scrad51 or the Sprad51 mutant. Therefore, a region between the 35th and 74th amino acids of the ScRad51 protein is probably involved in the complementation of MMS sensitivity of the Sprad51 mutant, while the region from the N-terminus to the 35th amino acid of the ScRad51 protein is also required for the ScRad51 function. The HsRAD51 gene does not have the region required to complement the defects in both Scrad51 and Sprad51 mutants. We found that the Scrad52-AK28.1 gene, that specifies a truncated protein missing 176 amino acid residues from the C-terminus (one-third of the protein), can complement the MMS sensitivity of the Scrad52-1 missense mutant that has a substitution at the 86th amino acid residue (8). However, this gene cannot complement the MMS sensitivity of Scrad52 deletion mutant (22, and our unpublished results). These results show that the C-terminal region of Rad52-1 mutant protein can suppress the deficiency of Rad52-AK28.1 protein. Similarly, the region containing the 86th amino acid of the Rad52AK28.1 protein can suppress the deficiency of Rad52-1 protein. The human Rad52 protein has no homology to the ScRad52 protein in one-half of the C-terminal region, but has 65% identical amino acids at the N-terminal half. The protein cannot complement the MMS sensitivity in either rad52-1 missense or Fad52 deletion mutants of S. cerevisiae (data not shown). Therefore, the human protein cannot be substituted for the yeast protein in these Scvad52 mutants. Because the presence of the physical interaction between RadSl and Rad.52 proteins in ok showed that the C-terminal one-third of Rad52 protein interacts with Rad51 protein (Ih), the deficiency of the suppression of Scrad52-1 mutant by the human Rad52 protein may result from failure of interaction between ScRad51 and HsRad52 proteins. The N-terminal region of the Rad51 protein and one-third of the C-terminal region of the Rad52 protein probably interact in a speciesspecific manner.

100

T.

OGAWA

ET

AL.

SUMMARY

The structures and properties of the Rad.51 and Rad52 proteins eukaryotes are described. Both proteins form a complex and are sponsible for recombination and repair reactions. The N-terminal gion of the Rad51 protein interacts with the C-terminal region of Rad52 protein. Species-specific interaction is probably essential for functioning of these genes.

in rerethe the

Acknowledgments We thank Dr. J. Tomizawa for critical reading of the manuscript. This work was supported in part by a Grant-in Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan, and by a Collaboration Research Grant from the National Institute of Genetics. REFERENCES 1 G. M. Clark and A. N. Margulies, Proc. Natl. Acad. Sci., U.S.A., 53, 451 (1965). 2 J. Tomisawa and H. Ogawa, Cold Spring Harbor Symp. Quant. Biol., 33, 243 (1968). 3 H. Ogawa and T. Ogawa, Adw. Biophys., 26, 33 (1990). 4 S.C. Kowalczykowski, D.A. Dixon, A.K. Eggleston, S.D. Lauder, and W.M. Rehrauer, J. Microbial. Rev., 58, 401 (1994). 5 S. C. Kowalczykowski and A. K. Eggleston, Annu. Rev. Biochem., 63, 991 (1994). 6 J. C. Game and R. K. Mortimer, Mutat. Res., 24, 281 (1974). 7 A. Shinohara, H. Ogawa, and T. Ogawa, Cell, 69, 457 (1992). 8 K. Adzuma, T. Ogawa, and H. Ogawa, Mol. Cell. Biol., 4, 2735 (1984). 9 T. Ogawa, A. Shinohara, A. Nabetani, T. Ikeya, X. Yu, E. H. Egelman, and H. Ogawa, Cold Spring Harbor Symp. Quant. Biol., 58, 567 (1993). 10 A. Shinohara, H. Ogawa, Y. Matsuda, N. Ushio, K. Ikeo, and T. Ogawa, Nat. Genet., 4, 239 (1993). 11 J. Tomizawa and H. Ogawa, J. Mol. Biol., 30, 7 (1967). 12 T. Ogawa, X. Yu, A. Shinohara, and E. H. Egelman, Science, 256,1896 (1993). 13 S. West, Annu. Rev. Biochem., 61, 603 (1992). 14 E. H. Egelman, Curr. Opin. Struct. Biol., 3, 189 (1993). 15 P. Sung, Science, 265, 1241 (1994). 16 G. T. Milne and D. T. Weaver, Gene Dew., 9, 1755 (1993). 17 0. Bezzubova, A. Shinohara, R. G. Mueller, H. Ogawa, and J.-M. Buerstedde, Nucl. Acids Res., 21, 1577 (1993). 18 D. K. Bishop, D. Park, L. Xu, and N. Kleckner, Cell, 69,439 (1992). 19 T. Kobayashi, Y. Hotta, and S. Tabata, Mol. Gem Genet., 237, 225 (1993). 20 K. Ostermann, A. Lorentz, and H. Schmidt, Nucl. Acids Res., 21, 5940 (1993). 21 0. Bezzubova, H. Schmidt, K. Ostermann, W. D. Heyer, and J.-M. Buerstedde, Nucl. Acids Res., 21, 5945 (1993). 22 K. L. Boundy-Mills and D. M. Livingston, Genetics, 133, 39 (1993).