Effect of bacterial recA expression on DNA repair in the rad51 and rad52 mutants of Saccharomyces cerevisiae

recA expression on DNA repair in the

rad51 and

rad52 mutants of

Saccharomyces cerevisiae

M.A. Morais Jr. ¹, V. Vlcková ⁴, I. Fridrichová ³, M. Slaninová ⁴, J. Brozmanová ³ and J.A.P. Henriques ²

¹ Departamento de Genética, Universidade Federal de Pernambuco, Av. Moraes Rego, s/n 50670-901 Recife, PE, Brasil. E-mail: [e-mail].

² Centro de Biotecnologia Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500, Prédio 2A B1, Campus do Vale, 91501-970 Porto Alegre, RS, Brasil. E-mail: [e-mail]. Send correspondence to J.A.P.H.

³ Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, 812 32 Bratislava, Slovakia. E-mail: [e-mail].

⁴ Department of Genetics, Comenius University, 842 15 Bratislava, Slovakia.

ABSTRACT

Molecular and functional homology between yeast proteins pRad51 and pRad52 and Escherichia coli pRecA involved in recombinational DNA repair led us to investigate possible effects of recA gene expression on DNA repair in rad51 and rad52 mutants of Saccharomyces cerevisiae. The mutant cells were subjected to one of the following treatments: preincubation with 8-methoxypsoralen and subsequent irradiation with 360-nm ultraviolet (UVA) (8-MOP + UVA), irradiation with 254-nm UV light or treatment with methyl methane sulfonate (MMS). While recA expression did not repair lethal DNA lesions in mutant rad51, it was able to partially restore resistance to 8-MOP + UVA and MMS in rad52. Expression of recA could not complement the sensitivity of rad51rad52 double mutants, indicating that pRad51 may be essential for the repair-stimulating activity of pRecA in the rad52 mutant. Spontaneous mutagenesis was increased, and 8-MOP-photoinduced mutagenesis was decreased by the presence of pRecA in rad52, whereas pRecA decreased UV-induced mutagenesis in rad51. Thus, pRecA may function in yeast DNA repair either as a member of a protein complex or as an individual protein that binds to mutagen-damaged DNA.

INTRODUCTION

Despite its multiple activities in general recombination, biochemical analysis revealed that the primary role of E. coli pRecA is in the repair of DNA lesions (reviewed in Kowalczykowski et al., 1994). E. coli recA gene homologs are found in diverse organisms, from bacteriophage to human cells, indicating that mechanisms of homologous recombination have been conserved during evolution (Miller and Kokjohn, 1990; Thompson, 1996). In contrast, other functions of pRecA were transferred to several other proteins during evolution of eukaryotic cells (Shinohara et al., 1992; Zdzienicka, 1995).

Recombinational repair in the yeast Saccharomyces cerevisiae is under the control of genes from the RAD52 epistasis group, which is characterized by sensitivity to the radiomimetic chemical methyl methane sulfonate (MMS) (Prakash and Prakash, 1977), photoactivated psoralens (Henriques and Moustacchi, 1980), and an extreme sensitivity to X-rays in their mutants (Game and Mortimer, 1974). Mutations in the RAD52 group confer, to a varying degree, defects in spontaneous and induced mitotic and meiotic recombination, DNA double-strand break (DSB) repair, sporulation and spore viability, mating-type switching, post-replication repair and plasmid integration (reviewed in Friedberg, 1988; Petes et al., 1991). Among genes of the RAD52 group are RAD54, a putative ATPase, which belongs to a new gene family encoding DNA helicases (Emery et al., 1991; Schild et al., 1992), and the recA homologs RAD51, RAD55, RAD57 and DMC1 (Kans and Mortimer, 1991; Bishop et al., 1992; Shinohara et al., 1992; Lovett, 1994). Biochemical characterization showed that pRad51 is a 43-kDa protein, which, like pRecA, forms a filament on DNA and catalyzes strand exchange in the presence of ATP (Ogawa et al., 1993a; Sung, 1994). The central role of RAD52 in genetic recombination and DSB repair is still not understood in biochemical terms: pRad52 is a 52.4-kDa protein that has no homology with either pRecA, nor with any known protein (Adzuma et al., 1984). A rad52 mutant has about 90% reduction in the level of nuclease RhoNuc (Terry et al., 1988). Genetic studies suggest that pRad52 is not required for initiation of recombination, but is essential for the intermediate step between formation of double-strand breaks and emergence of stable recombinants (Petes et al., 1991). pRad52 exhibits pRecA-like DNA strand transfer activity, though its efficiency is only 10% of that of pRecA (Ogawa et al., 1993b). pRad52 and pRad51 interact physically (Milne and Weaver, 1993) in a process essential for the majority of recombinational reactions.

In this communication we try to indirectly characterize the recA-like activities of proteins encoded in RAD51 and RAD52. We used pRecA as a probe for complementation of the high sensitivity of the corresponding rad51 and rad52 mutants to 8-MOP + UVA, UV-irradiation and MMS exposure. We showed that pRecA increases resistance of the rad52 mutant to 8-MOP photoaddition and MMS, and that it changes the mutagenic responses of both rad51 and rad52 mutants.

MATERIAL AND METHODS

Strains and plasmid

The strains of S. cerevisiae used in this study and their genotypes are listed in Table I. The haploid rad51 and rad52 disruption mutants and their isogenic wild types (WT) were kindly provided by F. Fabre. The haploid rad51 rad52 double mutant was produced by crossing and tetrad dissection (Rose et al., 1990). Bacterial strain DH5a was used for plasmid propagation (Sambrook et al., 1989).

StrainGenotypeSourceFF18733MATa leu2-3/112 lys1-1 his7-2 trp1-289 ura3-52F. FabreFF18742FF18733 rad52::URA3F. FabreFF18984MATa leu2-3/112 lys2-1 his7-1 ura3-52F. FabreFF18987FF18984 rad51::URA3F. FabreFF18985MATa leu2-3/112 lys2-1 his7-1 ura3-52F. FabreFF18988FF18985 rad51::URA3F. FabreMM06-7cMATa leu2-3/112 lys1-1 ade2-1 ura3-52 rad52::URA3This workMM08-4bMATa leu2-3/112 lys1-1 ade2-1 ura3-52 rad51::URA3This workMM10-2aMATa leu2-3/112 lys1-1 his7-2 trp1-289 ura3-52This workMM10-2cMATa leu2-3/112 lys1-1 his7-1 ura3-52 rad51::URA3 rad52::URA3This work

Strain	Genotype	Source
FF18733	MATa leu2-3/112 lys1-1 his7-2 trp1-289 ura3-52	F. Fabre
FF18742	FF18733 rad52::URA3	F. Fabre
FF18984	MATa leu2-3/112 lys2-1 his7-1 ura3-52	F. Fabre
FF18987	FF18984 rad51::URA3	F. Fabre
FF18985	MATa leu2-3/112 lys2-1 his7-1 ura3-52	F. Fabre
FF18988	FF18985 rad51::URA3	F. Fabre
MM06-7c	MATa leu2-3/112 lys1-1 ade2-1 ura3-52 rad52::URA3	This work
MM08-4b	MATa leu2-3/112 lys1-1 ade2-1 ura3-52 rad51::URA3	This work
MM10-2a	MATa leu2-3/112 lys1-1 his7-2 trp1-289 ura3-52	This work
MM10-2c	MATa leu2-3/112 lys1-1 his7-1 ura3-52 rad51::URA3 rad52::URA3	This work

Table I - Strains of S. Cerevisiae.

Construction of pVTLrecA has been described by Morais Jr. et al. (1994). The recA gene was inserted under the control of the ADH1 gene promoter on the multicopy plasmid pVT103L kindly provided by T. Vernet (Vernet et al., 1987). Yeast transformation was conducted by incubating yeast cells in rich medium before plating (Schiestl and Gietz, 1989).

Expression of RecA

Yeast extracts were prepared by sonication in the presence of a protease inhibitor cocktail (antipain, aprotinin, leupeptin, pepstatin A, and chymostatin) according to Cernaková et al. (1991). Anti-RecA polyclonal antibodies were prepared as described previously (Fridrichová et al., 1992). The extracts were analyzed by immunoblotting using peroxidase-labeled swine antirabbit IgG conjugates and diaminobenzydine, according to standard methods (Harlow and Lane, 1988; Sambrook et al., 1989).

DNA damage treatments

Cells were grown to exponential growth phase (1-2 x 10⁷ cells/ml) in the synthetic medium lacking leucine (SC-Leu) and, after washing, resuspended in sterile saline at the same concentration and subjected to one of the following treatments:

a) 8-MOP was added to 50 mM, and, after incubation of the suspension for 15 min at 4^oC, irradiated with 365-nm UVA in a Petri dish using a 9/7 W Osram lamp at a fluence rate of 1 kJ/m²/min.

b) Cells were exposed to 0.05% MMS for varying periods of time, and MMS was removed by washing the cells before plating.

c) The cell suspension was 254-nm UV irradiated in a Petri dish with a 15-W Philips TUV germicidal lamp at a fluence rate of 37.5 J/m² /min.

After appropriate dilution, cells were plated on YEPD (1% yeast extract, 2% peptone, 2% dextrose) for survival. For mutagenesis measurement, 100-300 m l of undiluted cell suspensions was plated on synthetic complete medium lacking histidine or tryptophan. The results shown are from three independent experiments with triplicate platings at each dose.

RESULTS

Expression of the recA gene

Cell-free extracts of rad51 and rad52 and the corresponding WT, transformed with pVTLrecA, were submitted to electrophoretic separation, and the RecA protein was detected by immunoblot assay. Figure 1 shows that yeast cells contain a 38-kDa protein corresponding to E. coli pRecA, indicating correct expression of recA in all yeast transformants.

Figure 1
- Identification of the RecA protein in protein extracts of yeast cells. Lane 1, Molecular weight standard; Lane 2, FF18733/pVT103L; Lane 3, FF18733/pVTLrecA; Lane 4, FF18742/pVT103L; Lane 5, FF18742/pVTLrecA; Lane 6, E. coli RecA protein; Lane 7, FF18984/pVT103L; Lane 8, FF18984/pVTLrecA; Lane 9, FF18987/pVT103L; Lane 10, FF18987/pVTLrecA; Lane 11, E. coli RecA protein.

Effect of the recA on cell survival

Survival after treatment with 8-MOP + UVA, UV, and MMS of the three yeast transformants and their corresponding non-transformed controls is shown in Figure 2. pRecA had no influence on WT survival after 8-MOP + UVA (Figure 2, panels A and D) and UV (Figure 2, panels C and F). The same was observed for rad51, though this mutant was more sensitive to all treatments (Figure 2, upper panels). This means that, albeit homologous, pRecA cannot complement the defect in DNA repair caused by a non-functional pRad51.

Figure 2 - Effect of the recA gene on survival of rad51 (upper panels) and rad52 (lower panels) mutants after 8-MOP plus UVA (A and D), MMS (B and E) and UV light (C and F) treatments. Symbols are wild types ( O ), rad51 ( ) rad52 ( D ) cells transformed with a plasmid containing (closed symbols) or not (open symbols) the recA gene.

Compared to rad51 cells, rad52 cells were even more sensitive to 8-MOP + UVA and MMS (Figure 2, lower panels). Expression of the recA gene was able to partially, but significantly, restore some resistance in rad52 to these two mutagens, and the transformant reached approximately the sensitivity level of the rad51 mutant (Figure 2, panels D and E). No effect of pRecA in the rad52 mutant was seen after UV irradiation (Figure 2, panel F). Therefore, pRecA can substitute some of the functional DNA repair activities lost in rad52.

When tested in a haploid rad51rad52 double mutant, pRecA could not complement the 8-MOP + UVA sensitivity phenotype (Figure 3). Thus, pRad51 needs to be functional for pRecA to carry out its sensitivity-complementing effect, as was shown by increased survival after 8-MOP + UVA in a recA- transformed rad52 mutant.

Figure 3 - Effect of the recA gene on survival of the rad51 rad52 double mutant after 8-MOP. Symbols are wild types ( O ) and mutant ( ) cells transformed with a plasmid containing (closed symbols) or not (open symbols) the recA gene.

pRecA influences spontaneous and induced mutation in yeast

The effect of pRecA on spontaneous and induced mutagenesis was tested in the three yeast strains. UV-induced mutagenesis was always higher than that induced by 8-MOP + UVA (Figure 4). The presence of pRecA increased the frequency of induced mutagenesis in WT after 8-MOP + UVA (Figure 4, panel A) and after UV (Figure 4, panel B). The frequency of induced mutation in rad51 (Figure 4, middle panels) and rad52 (Figure 4, lower panels) was very high after both treatments when compared to that of the WT. pRecA did not change the frequency of revertants in rad51 after 8-MOP + UVA (Figure 4, panel C), but lowered mutagenesis after UV (Figure 4, panel D). Also, pRecA reduced the frequency of induced reversion in rad52 after both treatments (Figure 4, panels E and F), mainly at higher mutagen doses. On the other hand, a three-fold increase in spontaneous mutagenesis was observed in rad52 transformants (data not shown).

Figure 4
- Effect of the recA gene on mutagenesis of the wild type (A and B), rad51 (C and D) and rad52 (E and F) strains after 8-MOP plus UVA (left panels) and 254-nm UV (right panels) treatments. Symbols are as in Figure 2.

DISCUSSION

Genetical and molecular characterization of the yeast genes RAD51 and RAD52 suggest that both have important functions in either general recombination or recombinational DNA repair. While pRad51 was shown to have molecular and functional homology with E. coli pRecA, the exact function(s) of the pRad52 is not so well defined. By heterologous overexpression of pRecA in yeast rad51 and rad52 mutants, we hoped to gain insight into a possible pRecA-like function of pRad51 and/or pRad52.

Our results show that pRecA, although sharing great homology to pRad51, failed to complement sensitivity to 8-MOP + UVA, UV and MMS of the rad51 mutant. The N-terminal region of pRad51, involved in ATP-dependent homologous pairing and strand exchange activities, was shown to be important for interaction between pRad51 and pRad52 (Shinohara et al., 1992; Milne and Weaver, 1993; Sung, 1994). Sequence alignment analysis revealed that 55 N-terminal amino acid residues of pRad51 from S. cerevisiae are not present in E. coli pRecA nor in human and mouse pRad51 homologs (Shinohara et al., 1993). Our results show that pRecA was not able to complement the repair defect of rad51 (Figure 2); the same was observed for the mammalian pRad51 homologs (Shinohara et al., 1993). Therefore, we propose that the bacterial pRecA, as well as mammalian pRad51 homologs, are not able to form an active complex with pRad52, since they lack the N-terminal region. Alternatively, the observed lack of complementation of sensitivity of the rad51 mutant by pRecA could be explained by the different polarity of DNA strand transfer reaction mediated by pRecA and pRad51 (Sung and Robberson, 1995).

The Rad52 protein, although not homologous to pRecA, seems to have some RecA-like biochemical functions. It is a DNA binding protein and promotes DNA strand transfer between homologous DNA molecules (Ogawa et al., 1993b; Mortensen et al., 1996). Recently it was found that overexpression of pRad52 in human somatic cells stimulates recombinational DNA repair (Johnson et al., 1996), and that expression of the human homolog of pRad52 increases the resistance of cultured monkey cells to ionizing radiation (Park, 1995). All this supports our speculation, that pRad52 could have conserved some functions similar to those of the E. coli pRecA, that is involved in DSB repair. Previously, we did not observe any complementation of resistance in the rad52-1 point mutant to gamma-irradiation by overexpression of the E. coli pRecA (Brozmanová et al., 1991). The present results show that pRecA can partially complement sensitivity of the rad52:URA3 disruption mutant to 8-MOP + UVA and MMS (Figure 2). Moreover, inhibition of cell growth by pRecA overexpression was observed in rad52:URA3 (data not shown), but not in rad52-1. This could mean that, in the absence of pRad52, pRecA would non-specifically interact with pRad51, thereby increasing cell resistance but interfering with DNA metabolism. In light of the genetic complexity of recombinational DNA repair in yeast, it seems likely that pRecA could function in yeast cells with assistance of the yeast recombination proteins. Recently Hays et al. (1995) demonstrated that DSB repair in yeast is catalyzed by a so-called recombinosome complex. Our results would thus predict that pRecA can substitute pRad52 in this complex and interact with pRad51. Whether protein-protein interaction between heterologous bacterial RecA and yeast Rad51 proteins occurs remains to be elucidated. This kind of interaction is indirectly supported by the fact that in the absence of pRad51, pRecA failed to restore mutagen resistance in rad52 (Figure 3). Moreover, yeast y-RPA protein was able, at least in an in vitro system, to stimulate the DNA strand exchange activity of pRecA protein to the same degree as it was observed with the yeast Sep1 protein, thus indicating heterologous interaction between the y-RPA and RecA proteins (Alani et al., 1992).

rad51 and rad52 mutants had more spontaneous mutagenesis than the WT, as first reported for rad52 by von Borstel et al. (1973). Both mutants displayed hypermutability after 8-MOP + UVA and UV (Figure 4; Morais Jr. et al., 1996). This indicates that repair of DNA lesions is deviated to an error-prone pathway in these recombination-deficient mutants and implicates a function of RAD51 and RAD52 in cellular processes other than DSB repair (Resnick et al., 1995). Rattray and Symington (1994) suggested that initiated recombination events in rad52 can be rescued by an error-prone pathway in this mutant. The expression of recA increased UV-induced mutagenesis in WT (Figure 4), confirming earlier results (Brozmanová et al., 1991). It also changed the mutagenic response in both mutants. A three-fold increase in spontaneous mutagenesis was observed in rad52 (data not shown), whereas the frequency of induced revertants decreased after high doses of 8-MOP + UVA and UV (in rad51 only after UV). Also, pRecA could restore the deficiency of the pso4-1 mutant to 8-MOP + UVA-induced mutagenesis (Morais Jr. et al., 1994). This demonstrates that overexpressed pRecA can produce different responses in yeast mutants that have particular defects in recombinational DNA repair; different responses might be elicited by channeling DNA lesions to either error-free or error-prone repair pathways. The mechanisms of these effects remain to be elucidated.

ACKNOWLEDGMENTS

We thank Dr. Francis Fabre for providing strains and Dr. Martin Brendel and J.F. dos Santos for critically reading the manuscript. This work was supported by the Brazilian agencies: Programa de Formação de Recursos Humanos em Áreas Estratégicas (RHAE), Fundação de Apoio à Pesquisa no Estado do Rio Grande do Sul (FAPERGS) and Genotox Laboratory/CBIOT/UFRGS, as well as the Slovak Grant Agency for Science (grants Nos. 2/1330/94 and 1/1155/94).

RESUMO

A homologia tanto a nível molecular como funcional entre as proteínas de leveduras pRad51 e pRad52 envolvidas na reparação de DNA tipo recombinacional e pRecA de E. coli nos levou a analisar os possíveis efeitos da expressão do gene recA sobre a reparação de DNA nos mutantes rad51 e rad52 de S. cerevisiae após tratamento com 8-MOP + UVA, com UV e com MMS. A expressão de recA não foi capaz de restaurar a reparação das lesões induzidas no DNA do mutante rad51 após tratamento com esses agentes, entretanto ela restaurou parcialmente a resistência ao 8-MOP + UVA e ao MMS no mutante rad52. A expressão de recA não complementou a sensibilidade do duplo mutante rad51rad52, indicando que pRad51 pode ser essencial para estimular a atividade de reparação da pRecA no mutante rad52. A presença de pRecA no mutante rad52 aumentou a mutagênese espontânea e reduziu a mutagênese fotoinduzida pelo 8-MOP, enquanto que a pRecA diminuiu a mutagênese induzida pela UV no mutante rad51. Conseqüentemente, no reparo de DNA em levedura, a pRecA pode funcionar tanto como membro de um complexo protéico ou como uma proteína individual que se liga à lesão no DNA provocada pelo agente mutagênico.

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(Received March 12, 1997)

References

• Adzuma, K., Ogawa, T. and Ogawa, H. (1984). Primary structure of the RAD52 gene in Saccharomyces cerevisiae. Mol. Cell. Biol. 4: 2735-2744.
• Alani, E., Thresher, R., Griffith, J.D. and Kolodner,R.D. (1992). Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand DNA-binding protein. J. Mol. Biol. 227: 54-71.
• Bishop, D.K., Park, D., Xu, L. and Kleckner,N. (1992). DMC1: a meiose-specific yeast homolog of E. coli RecA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell 69: 439-456.
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Publication Dates

• Publication in this collection
  06 Jan 1999
• Date of issue
  Mar 1998

History

• Received
  12 Mar 1997