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As a consequence, bacterial DNA repair genes encoding proteins with well ... DNA repair Heterologous expression Saccharomyces cerevisiae Escherichia coli.
Curr Genet (2004) 46: 317–330 DOI 10.1007/s00294-004-0536-2

R EV IE W A RT I C L E

Jela Brozmanova´ Æ Viera Vlcˇkova´ Æ Miroslav Chovanec

How heterologously expressed Escherichia coli genes contribute to understanding DNA repair processes in Saccharomyces cerevisiae

Received: 16 July 2004 / Revised: 13 September 2004 / Accepted: 18 September 2004 / Published online: 13 November 2004  Springer-Verlag 2004

Abstract DNA-damaging agents constantly challenge cellular DNA; and efficient DNA repair is therefore essential to maintain genome stability and cell viability. Several DNA repair mechanisms have evolved and these have been shown to be highly conserved from bacteria to man. DNA repair studies were originally initiated in very simple organisms such as Escherichia coli and Saccharomyces cerevisiae, bacteria being the best understood organism to date. As a consequence, bacterial DNA repair genes encoding proteins with well characterized functions have been transferred into higher organisms in order to increase repair capacity, or to complement repair defects, in heterologous cells. While indicating the contribution of these repair functions to protection against the genotoxic effects of DNAdamaging agents, heterologous expression studies also highlighted the role of the DNA lesions that are substrates for such processes. In addition, bacterial DNA repair-like functions could be identified in higher organisms using this approach. We heterologously expressed three well characterized E. coli repair genes in S. cerevisiae cells of different genetic backgrounds: (1) the ada gene encoding O6-methylguanine DNA-methyltransferase, a protein involved in the repair of alkylation damage to DNA, (2) the recA gene encoding the main recombinase in E. coli and (3) the nth gene, the product of which (endonuclease III) is responsible for the repair of oxidative base damage. Here, we summarize our results and indicate the possible implications

Communicated by S. Hohmann J. Brozmanova´ (&) Æ M. Chovanec Laboratory of Molecular Genetics, Cancer Research Institute, Vla´rska 7, 833 91 Bratislava, Slovak Republic E-mail: [email protected] Tel.: +421-2-59327333 Fax: +421-2-59327350 V. Vlcˇkova´ Faculty of Natural Sciences, Department of Genetics, Comenius University, 842 15 Bratislava, Slovak Republic

they have for a better understanding of particular DNA repair processes in S. cerevisiae. Keywords DNA repair Æ Heterologous expression Æ Saccharomyces cerevisiae Æ Escherichia coli

Introduction The integrity of DNA inside cells is constantly being challenged by endogenous and exogenous DNA-damaging agents. The forms of DNA damage inflicted are many and include single- and double-strand breaks (SSB, DSB), chemically modified bases, abasic sites, inter- and intra-strand cross-links and mismatched base pairs. To maintain the integrity of the genome, living organisms have evolved a variety of systems that recognize and repair different forms of DNA damage. The field of DNA repair has made considerable progress over the past 40 years. It has become evident that DNA repair is tightly connected with basic cellular processes, such as transcription and replication. Defects in DNA repair pathways provide the molecular basis of genetic diseases associated with sensitivity to DNA-damaging agents and, in multicellular organisms, can initiate events that result in cancer (Friedberg 2000; Hoeijmakers 2001). The lower eukaryotic organism, the budding yeast Saccharomyces cerevisiae, has already proven to be a powerful eukaryotic model system for DNA repair studies on a molecular and cellular level. The results obtained using S. cerevisiae led in many cases to the discovery of several important repair phenomena and pathways and turned them into highly relevant areas of investigation in human biology and diseases (Game 2000; Resnick and Cox 2000). Systematic research of mutagen–cell interaction in S. cerevisiae led to the discovery of great number of genetic loci with a function in DNA repair. Yeast mutants originally isolated by virtue of their radiation

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sensitivity have been organized into three epistasis groups, designated RAD3, RAD6 and RAD52, representing the different systems of DNA repair. Phenotypic characterization of these mutants indicated that the RAD3 epistasis group comprises genes involved in nucleotide excision repair. Mutants in the RAD52 epistasis group are defective in genetic recombination and in the repair of DNA strand breaks; and these genes are therefore believed to be required for recombinational DNA repair. Many of the genes in the RAD6 epistasis group are required for spontaneous and/or damageinduced mutagenesis, suggesting that they participate in biochemical events associated with altered replication fidelity (Friedberg 1988, 1991; Friedberg et al. 1992). Despite the heuristic value of this classification, it should be noted that the epistasis pathways are not mutually exclusive. Some loci appear to belong simultaneously to two epistasis pathways and members of two or more pathways can have various properties in common (Roche et al. 1993). However, several genes are outside the three original groups, and therefore they have not been designated as RAD loci (Game 2000). There are additional genes, e.g. the PSO genes encoding proteins involved in the repair of DNA lesions induced by monoand bi-functional psoralens (Henriques and Brendel 1990; Henriques et al. 1997), which have been added to the epistasis groups on the base of phenotypic similarities to mutant strains already tested. The pso mutants have phenotypically been characterized and some of the PSO genes have been identified and cloned (for reviews, see Brendel and Henriques 2001; Brendel et al. 2003). Other genes that are not included in the three epistasis groups are those encoding photoreactivation enzyme, base excision repair (BER) factors, mismatch repair (MMR) factors and those operating in DSB repair by non-homologous end-joining. DNA repair processes have been shown to have a high degree of evolutionary conservation from prokaryotic to eukaryotic organisms (Jazayeri and Jackson 2002). This allows the horizontal transfer of genes that encode DNA repair proteins from lower organisms in order to investigate DNA repair processes in more complex organisms. For example, using appropriate shuttle vectors, DNA repair genes from Escherichia coli can be expressed in the lower eukaryote S. cerevisiae, either to provide increased capacity to repair corresponding lesions in repair-competent cells, or to complement repair defects in repair-deficient mutants. Several years ago, we and our collaborators started experiments focused on finding E. coli-like repair activities in the yeast S. cerevisiae, using a complementation approach. In the course of these studies, three well characterized E. coli DNA repair genes, each acting by a different mechanism in the cellular response to DNA damage, were heterologously expressed in S. cerevisiae. These were: (1) the ada gene encoding O6-methylguanine DNA-methyltransferase (MTase), a protein involved in the repair of alkylation damage to DNA by mechanism known as DNA damage reversal, (2) the recA gene

encoding the main recombinase in E. coli that acts in the process of DNA damage tolerance and (3) the nth gene, the product of which (endonuclease III; endo III), is responsible for the repair of oxidative base damage by a BER mechanism. A number of heterologous expression studies had been reported from the middle 1980s to the early 1990s. These showed that the T4 denV gene product, endonuclease V, complemented ultraviolet (UV) sensitivity of the S. cerevisiae rad1 and rad3 mutants (Chenevert et al. 1986; Valerie et al. 1986) and restored colony-forming ability and excision repair synthesis after UV irradiation in human XP cells (Valerie et al. 1987). In addition, the heterologously expressed E. coli phr gene, encoding a photolyase, complemented photoreactivation activity in photoreactivation-deficient S. cerevisiae mutants (Langeveld et al. 1985). Earlier heterologous expression studies of the E. coli ada and nth genes showed that the ada gene increased cellular resistance to alkylation agents in repair-deficient human cells (Samson et al. 1986; Brennand and Margison 1986) and the nth gene conferred resistance to hydrogen peroxide (H2O2) and bleomycin in gamma-ray sensitive Chinese hamster ovary cells (Harrison et al. 1992). Here, we summarize the findings of our heterologous expression experiments and their implications in the understanding of DNA repair processes in S. cerevisiae.

Heterologous expression of the ada gene Alkylating agents are a large group of environmentally relevant compounds that can cause a variety of biological effects, including toxicity, mutagenicity and, ultimately, malignant transformation in multicellular organisms. These effects are mainly attributed to the production of alkylation damage in DNA (Saffhill et al. 1985). Several sites in DNA can be alkylated, the N7 position of guanine being the most frequent (Singer 1975; Beranek et al. 1980). Perhaps the most relevant adduct is, however, O6-methylguanine (O6-MeG), which is the principal mutagenic and toxic DNA lesion induced by methylating agents such as N-methyl-N¢-nitro-Nnitrosoguanidine (MNNG). The mutagenic and toxic potential of O6-MeG resides in its ability to mispair with thymine during DNA replication, resulting in G:C to A:T transition mutations (for reviews, see Saffhill et al. 1985; Lindahl et al. 1988; Kleibl 2002). The cytotoxic effect of O6-MeG is mediated by the post-replication MMR system (Kat et al. 1993). In E. coli, two main groups of repair factors have evolved to deal specifically with alkylation damage to DNA: one is inducible (also referred to as the adaptive response) and the other is constitutive (for reviews, see Lindahl et al. 1988; Kleibl 2002). The adaptive response, originally described by Jimenez-Sanchez and CerdaOlmedo (1975) and Samson and Cairns (1977), is induced by sublethal, non-mutagenic doses of alkylating

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agents. This leads to a higher resistance of pretreated cells to a challenge dose of these agents, compared with non-pretreated cells. This is a consequence of induced expression of the ada, alkA, alkB and aidB genes. The constitutive genes are ogt (Potter et al. 1987) and tag (Clarke et al. 1984), the products of which are assumed to be the constitutive homologues of Ada and AlkA, respectively (for reviews, see Lindahl et al. 1988; Kleibl 2002). The Ada protein has a regulatory function in the adaptive response. It is a dual MTase that transfers methyl groups from O6-MeG, O4-methylthymine (O4MeT) and methylphosphotriesters (MePTs) in DNA to cysteine acceptor sites (Cys 69, 321) in the protein itself. Cys 321 acquires the methyl groups from either O6-MeG or O4-MeT, while Cys 69 is the acceptor site for the S-diastereoisomer of the two possible MePT adducts (Lindahl et al. 1988; Sedgwick et al. 1988; Kleibl 2002). Methyl transfer from MePT to Cys 69 converts the Ada protein from a weak to a strong transcriptional activator that binds to a common regulatory DNA sequence upstream of the ada-alkB operon and the alkA gene. Ada also up-regulates the aidB gene, but the mechanism is not known. The AlkA protein is 3-methyladenine DNA glycosylase II that repairs 3-methyladenine (3-MeA), 3-methylguanine, O2methylguanine and O2-methylthymine DNA lesions. Since 3-methylpurines are assumed to be the main DNA replication-blocking lesions, AlkA protects against alkylation-induced cell death. The mechanism of the AlkB protein was only recently revealed and comprises an oxidative demethylation of 1-methyladenine and 3-methylcytosine adducts (Falnes et al. 2002; Trewick et al. 2002; Aas et al. 2003). No clear function has yet been assigned to AidB. Because of the occurrence of MTases and the adaptive response in a wide variety of microbes (for a review, see Samson 1992), from the middle to the late 1980s several studies investigated alkylation adaptation and repair in S. cerevisiae. These studies used two different approaches, either determining the toxic and mutagenic effects of high challenging doses of MNNG applied to cells pretreated with low doses of the same agent, or determining the rate of removal of alkylated bases from DNA by yeast cell extracts. However, these experiments showed the lack of any adaptive response in S. cerevisiae: growth of yeast cells in low concentrations of MNNG did not increase their resistance to killing and mutagenesis by subsequent high challenge doses of the agent (Maga and McEntee 1985; Polakowska et al. 1986). Moreover, they disclosed a defect of yeast cell extracts in their ability to remove alkylated bases from DNA (Maga and McEntee 1985; Goth-Goldstein and Johnson 1990). Taken together, these results suggested that alternative repair pathways might operate in S. cerevisiae to deal with alkylation damage to DNA. However, this notion turned out to be incorrect. In 1990, the first evidence was produced that the processes acting on alkylated DNA exist in S. cerevisiae. First,

Goth-Goldstein and Johnson (1990) reported that 7-methylguanine, 3-MeA and O6-MeG adducts can be efficiently removed from genomic DNA in S. cerevisiae. Second, Sassanfar and Samson (1990) showed that S. cerevisiae cell extracts could transfer methyl groups irreversibly from O6-MeG in DNA to a 25-kDa protein. This protein, the yeast MTase, was expressed at approximately 150 molecules per exponentially growing cell, but was undetectable in stationary cells. Surprisingly, the S. cerevisiae MTase was very temperaturesensitive, having a half-life of about 4 min at 37C; and this was presumably the reason why that others had previously failed to detect it. The yeast MTase was not inducible by low doses of alkylating agents, but rather was depleted in exposed cells, which was in line with previous in vivo results (Maga and McEntee 1985; Goth-Goldstein and Johnson 1990). Further biochemical experiments showed that the S. cerevisiae MTase could also be inactivated by O4-MeT (Sassanfar et al. 1991). Based on its molecular mass and substrate specificity, the S. cerevisiae MTase was proposed to be more closely related to mammalian than to E. coli MTases. At the same time, our laboratory was also focused on the repair of DNA alkylation damage in S. cerevisiae. We used an approach that was based on heterologous expression of the E. coli ada gene in this lower eukaryote. This was achieved by cloning the E. coli ada gene protein-coding region into an extrachromosomally replicating yeast expression vector (pADH) downstream of the constitutive promoter to yield pADHO6C (Brozmanova´ et al. 1990). Three different wild-type S. cerevisiae strains were transformed with pADH or pADHO6C. As expected, pADHO6C transformation resulted in high-level expression of the ada-encoded MTase, whereas pADH tranformants contained virtually no activity. High levels of Ada MTase resulted in a substantial increase in resistance to the toxic and mutagenic effects of MNNG (Fig. 1, Table 1). Our results therefore showed that the MTase-mediated demethylation repair pathway, despite its bacterial origin, can operate in S. cerevisiae to repair alkylated bases in DNA. Furthermore, they suggested that the same activity might exist in S. cerevisiae. We were also one of the laboratories that investigated the possibility that S. cerevisiae may contain an adaptive response analogous to that in E. coli. Consistent with other reports (Maga and McEntee 1985; Polakowska et al. 1986), we provided evidence that no such response occurs in S. cerevisiae (Brozmanova´ et al. 1990). In 1991, the S. cerevisiae MTase gene, MGT1, was cloned and the role of its product in protecting yeast cells from alkylation damage to DNA in vivo was determined (Xiao et al. 1991). The MGT1 gene was isolated by screening a library for a yeast DNA fragment that was able to suppress alkylation-induced killing and mutation in an E. coli ada ogt deficient mutant. The cloned DNA fragment contained an open reading frame, designated MGT1, which encoded a 206-amino-acid protein with a molecular mass of 23.575 kDa (Xiao and

320 Fig. 1 Results obtained by heterologous expression of the E. coli ada gene in the yeast S. cerevisiae. Only results that gave either positive or negative results are presented

Table 1 Summary of heterologous expression studies considered in this review Gene Recipient yeast strain: expression system used expressed ada

nth

recA

a

Three wild-types of different origin: ada gene expressed from multicopy, extrachromosomally replicating plasmid Wild-type, rad1, rad6 and rad52 cells: ada gene expressed from multicopy, extrachromosomally replicating plasmids

Observed effects

References

Increased resistance of all wild-types to toxic and mutagenic effects of MNNGa

Brozmanova´ et al. (1990)

Increased resistance of rad1 and rad6 cells to toxic and mutagenic effects of MNNG No effect on protection againts MNNG in rad52 cells Two wild-types of different origin: ada gene Increased resistance of both wild-types to integrated into genomic DNA toxic and mutagenic effects of MNNG to level comparable with the multicopy plasmid system Increased resistance to toxic effects of CCNU, but not MMS Two wild-type and pso3 mutant strains of Recovery of survival, decreased mutability different origin: nth expressed from multicopy, and protection of DNA against doubleextrachromosomaly replicating plasmids strand breakage in pso3 cells after H2O2 treatment Wild-type and rad52 cells: nth expressed from Increased resistance to the toxic effects of IR, multicopy, extrachromosomaly replicating but not those of H2O2 in wild-type cells No effect on sensitivity to IR and H2O2 plasmids in rad52 cells Two wild-types of different origin and one rad52 Increased resistance of the wild-types to the strain isogenic to one of the wild-types: recA toxic effects of IR and UV radiation and expressed from multicopy, extrachromosomaly increased UV-induced mutagenesis replicating plasmids No effect on protection of rad52 cells againts IR Two wild-type and pso4 mutant strains of Increased resistance of pso4 cells to 8-MOP + different origin: recA gene expressed from UVA and restored induced mutability after multicopy, extrachromosomaly replicating treatment with 8-MOP + UVA, UV and plasmids MNNG Diploid wild-type and mutant homozygous for Increased UV-induced non-reciprocal (gene pso4: recA expressed from multicopy, conversion) mitotic recombination in extrachromosomaly replicating plasmids wild-type cells No effect on 8-MOP + UVA-, UV- and MNNG-induced reciprocal (crossing over) or non-reciprocal (gene conversion) mitotic recombination in pso4 cells Two wild-type and rad52 mutant strains of Partial increase in resistance to MMS, 8-MOP different origin and one rad51 mutant strain + UVA and IR and full complementation of isogenic with one of the wild-types and rad52 DSB repair defect after exposure to IR and strains: recA expressed from multicopy, MMS in rad52 cells extrachromosomaly replicating plasmids No complementation of sensitivity of rad51 strain to MMS and 8-MOP + UVA

Brozmanova´ et al. (1994)

Farkasˇ ova´ et al. (2000)

Brozmanova´ et al. (2001b)

Sˇkorvaga et al. (2003)

Brozmanova´ et al. (1991)

Morais et al. (1994) Slaninova´ et al. (1996) Vlcˇkova´ et al. (1994, 1997)

Morais et al. (1998) Duda´sˇ et al. (2003)

For abbreviations, see text

Samson 1992). As might have been predicted, the Mgt1 protein displayed extensive homology with bacterial and human DNA MTases, mgt1 mutants lacked MTase

activity and were very sensitive to the toxic and mutagenic effects of alkylating agents, and MGT1 transcript levels were not increased in response to DNA alkylation

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damage. Nor was the Mgt1 MTase involved in the regulation of the yeast 3-methyladenine DNA glycosylase gene. In the meantime, to assess the extent to which ada expression might complement different repair defects in S. cerevisiae, we expressed the E. coli ada gene in wildtype and rad1, rad6 and rad52 mutant cells (Brozmanova´ et al. 1994; Fig. 1, Table 1). We placed the E. coli ada gene under the control of the constitutive promoter in two extrachromosomally replicating plasmids, pADH and pVT103L, to produce recombinant ada-expressing plasmids, pADHO6C and pVT103LO6C, respectively. Empty and recombinant vectors were introduced into wild-type, rad1, rad6 and rad52 cells and the biological consequence of ada expression in generated transformants was examined. As before (Brozmanova´ et al. 1990), very high levels of Ada MTase were achieved in all pADHO6C or pVT103LO6C transformants (Brozmanova´ et al. 1994). In line with a previous report (Cooper and Waters 1987), rad1 mutation did not confer increased sensitivity to the toxic and mutagenic effects of MNNG. We also showed that the effect of ada expression in the rad1 mutant was similar to that in the wildtype cells, i.e. there was extensive protection against the toxic and mutagenic effects of MNNG in ada-expressing rad1 cells (Fig. 1, Table 1). We therefore suggested that the RAD1 gene product is not essential for the repair of MNNG-induced DNA lesions nor for the ada-mediated repair of such lesions. In contrast to rad1, the rad6 mutant was more sensitive to the lethal and mutagenic effects of MNNG than the wild-type cells. Since ada expression led to an increased resistance of this mutant to the toxic and mutagenic effects of MNNG (Fig. 1, Table 1) to the level comparable with that seen in the wild-type cells, it was concluded that Ada MTase can repair MNNG-induced lesions in yeast, even if Rad6 is not functional. Assuming that the endogenous Mgt1 MTase is expressed in the rad6 mutant cells, the increased sensitivity of these cells to MNNG may be a consequence of a requirement of Rad6, or other factors that are under the control of Rad6, for the complete functional activity of the yeast Mgt1 MTase (Brozmanova´ et al. 1994). The highest sensitivity to the genotoxic effects of MNNG was observed in rad52 mutant cells. In these cells, however, neither MNNG-induced cell death nor mutation frequency was affected by ada expression (Table 1). This suggested that Rad52 might play an important role in the repair of alkylated DNA. In addition, Rad52 or, by inference, homologous recombination (HR) is required for the complete functioning of the E. coli Ada MTase in the budding yeast. This was the first suggestion of a possible co-factor requirement for MTase in eukaryotic cells (Brozmanova´ et al. 1994). Since the interpretation of these results was potentially complicated by the likelihood that individual cells may express widely different levels of Ada MTase activity, we generated and characterized stable yeast clones in which the E. coli ada gene was integrated into genomic DNA (Farkasˇ ova´ et al. 2000). Predictably,

MTase activities in extracts of all integrative clones carrying the ada gene were significantly higher than in control strains. However, these levels were approximately ten times lower than those in multicopy episomal vector ada transformants. Nevertheless, as was case for multicopy episomal vector ada transformants, the increased Ada MTase levels in integrative clones correlated with considerable protection against the toxic and mutagenic effects of MNNG (Fig. 1, Table 1). The level of protection in integrative clones was comparable with that in multicopy episomal vector ada transformants, suggesting that yeast may be able to utilize only limited amounts of Ada MTase for the repair of DNA alkylation damage. An alternative explanation was that the levels of Ada MTase expressed in the episomal vector population might be widely heterogeneous, so that some cells may express very high levels of Ada MTase and be no more resistant than cells expressing much lower Ada MTase levels, but contribute disproportionately to the overall levels of MTase activity. In addition to protection against MNNG, yeast clones stably expressing ada displayed increased resistance to the toxic effects of 3cyclohexyl-1-chloroethylnitrosourea (CCNU; Fig. 1, Table 1). This indicates that the Ada protein can prevent lethal CCNU-induced cross-links either by repairing O6chloroethyl monoadducts or by complexing with the cyclic intermediate N1-O6-ethanoguanine. In contrast, there was a lack of protection against the toxic effects of methyl methanesulfonate (MMS) in ada-expressing stable clones (Table 1), which corresponds with the very low levels of O-alkylated lesions produced in DNA by this agent. In summary, our results supported the conclusion that there is no adaptive response to alkylation damage in S. cerevisiae. Furthermore, they provided evidence that a damage-reversal repair pathway per se can act on yeast DNA to repair O-alkylated lesions, but requires cofactors for its operation. Finally, they showed that the level of MTase, as indicated by measurements on cellfree extracts, does not necessarily correlate with the protection provided.

Heterologous expression of the recA gene HR is an essential biological process that involves the pairing and exchange of DNA between two homologous sequences. It is of fundamental importance to the preservation of genomic integrity, the production of genetic diversity and the proper segregation of chromosomes during meiosis. As they disrupt both DNA strands, DSB are potentially very deleterious; and in mitotic cells the primary function of HR is the repair of DSB (for reviews, see Symington 2002; van den Bosch et al. 2002; Duda´sˇ and Chovanec 2004; Aylon and Kupiec 2004). DSB arise frequently as a consequence of normal metabolism such as collapsed replication forks, or oxygen metabolism which can generate reactive free radicals. They can also be generated by a number of

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exogenous agents, such as ionizing radiation (IR) and some chemicals. Unrepaired DSB can cause loss of genome integrity and/or cell death. If repaired incorrectly, they can also cause genetic alterations that can lead to cancer in multicellular organisms. The E. coli recA gene encodes a protein which is important in a diverse group of DNA metabolic processes, including HR, efficient post-replication repair and expression of a coordinated cellular response to DNA damage known as the SOS response (Walker 1984). The pivotal role of the RecA protein in initiating recombination was discovered by Clark and his colleagues more than three decades ago (Clark and Margulies 1965) and the first biochemical activities of the protein were described in 1979 (Ogawa et al. 1979; Roberts et al. 1979). Extensive genetic and biochemical analyses subsequently revealed the structure and function of the RecA protein, which contains 352 amino acid residues and has a molecular mass of 37.8 kDa (for reviews, see Kowalczykowski et al. 1994; Roca and Cox 1997). RecA promotes the ATP- and single-stranded DNA (ssDNA)-dependent cleavage of the LexA repressor, leading to the induction of the SOS response (Little et al. 1980). In addition, it performs two reactions important for HR: ATP-stimulated DNA strandannealing between complementary single strands of DNA and ATP-dependent DNA strand-exchange between ssDNA and the homologous sequence within double-stranded DNA (dsDNA; for reviews, see Kowalczykowski et al. 1994; Roca and Cox 1997). Mutations in the recA gene are pleiotropic, affecting not only recombination, but also overall DNA repair, SOS mutagenesis and cell division. Despite its multiple activities, the primary and absolutely essential role of RecA is in DSB repair by HR. The RecA protein has been the subject of many studies and has become the prototypic protein with which all other DNA strandannealing and DNA strand-exchange proteins are compared (Camerini-Otero and Hsieh 1995; Karlin and Brocchieri 1996). Our experiments involved an assessment of RecA-like activities in S. cerevisiae by a complementation Fig. 2 Results obtained by heterologous expression of the E. coli recA gene in the yeast S. cerevisiae. Only results that gave either positive or negative results are presented

approach. At that time, no RecA homologues were known, either in yeast or in other eukaryotic organisms. As before, we introduced the protein-coding region of the E. coli recA gene into the extrachromosomally replicating yeast expression vector (pADH) downstream of the constitutive ADH1 promoter to produce pADH recA. RecA expression in yeast was easily verified by immunoblotting, using anti-RecA polyclonal antibodies generated in our laboratory (Cˇerna´kova´ et al. 1991; Fridrichova´ et al. 1992). In our early studies, the E. coli RecA protein was expressed in the wild-type and in recombination-defective rad52 mutant cells (Cˇerna´kova´ et al. 1991). We found that wild-type pADH recA transformants were more resistant to the toxic effects of IR and UV but had an increased level of UV-induced mutation frequency, compared with pADH transformants (Fig. 2, Table 1). In contrast, recA expression did not complement the extreme sensitivity of the rad52 mutant to IR (Table 1). Thus, the RecA protein provided S. cerevisiae cells with additional DNA repair activities, which were shown to be error-prone and Rad52-dependent (Brozmanova´ et al. 1991). Apart from the functions already mentioned (see text above), the E. coli RecA protein is also involved in induced mutagenesis on the transcriptional and posttranslational level of translesion DNA synthesis (TLS), roles that are connected with its co-protease activity. In one process, RecA as a co-protease helps LexA to autodigest, thereby inactivating its repressor function and leading to derepression of transcription of the SOSregulated genes, including umuD, umuC, dinB etc., the products of which participate in TLS. In another process, UmuD undergoes a RecA/ssDNA-facilitated autodigestion that is mechanistically similar to that of LexA. This post-translation modification serves to remove the N-terminal 24 residues of UmuD, yielding UmuD¢ (Shinagawa et al. 1988). The UmuD¢2 homodimer interacts with UmuC (the catalytic subunit of DNA polymerase V), forming the UmuD¢2C complex that functions as a lesion by-pass polymerase to enable TLS (Sutton et al. 2002). UmuD¢ interacts with the a-catalytic subunit of DNA polymerase III which is suggested to be

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important for TLS, perhaps as part of a polymeraseswitching mechanism (Sutton et al. 2002). Although the S. cerevisiae mutants belonging to the RAD52 epistasis group exhibit a recombination-defective phenotype similar to the bacterial recA mutant, they do not confer the non-mutable phenotype that is a typical feature of recA mutants. Thus, the genes of the RAD52 epistasis group do not seem to control error-prone repair in the budding yeast. However, a simultaneous deficiency in mutation induction and recombination was reported for the pso4 (psoralen-sensitive) mutant (de Andrade et al. 1989; Henriques et al. 1989; Henriques and Brendel 1990). This suggested a phenotypic similarity between the S. cerevisiae pso4 mutant and the E. coli recA mutant. Since phenotypic characterization of the pso4 mutant suggested that Pso4 could play a role in the error-prone pathway that involves a recombination mechanism (Henriques and Brendel 1990), we used the recA gene to complement the individual repair defects of the pso4 mutant. To achieve this, we cloned the E. coli recA gene into the extrachromosomally replicating yeast vectors pADH and pNF2 to produce pADH recA (containing the URA3 gene for selection) and pNF2recA (containing the kanamycin resistance gene for selection). We found that the RecA protein partially complemented the 8-methoxypsoralen + UVA (8-MOP + UVA) sensitivity of the pso4 mutant and restored its mutability almost to the wild-type level after 8-MOP + UVA, UV and MNNG treatments (Morais et al. 1994; Slaninova´ et al. 1996; Fig. 2, Table 1). This suggested that the Pso4 protein could have a RecA-like function in induced mutagenesis and that Pso4 and RecA, the two proteins with pleiotropic functions, could act in a similar manner with regard to TLS. Further confirmation of the role of Pso4 in induced mutagenesis was obtained using pso4 rad51 and pso4 rad52 double mutants, in which the hypermutability of the rad51 and rad52 single mutants was abolished by introducing a pso4 mutation (Morais et al. 1996). When expressed in pso4 diploid cells, RecA had no effect on reciprocal (crossing-over) and non-reciprocal (gene conversion) mitotic HR induced by 8-MOP + UVA, UV and MNNG (Vlcˇkova´ et al. 1997; Table 1). Thus, RecA was not able to substitute for Pso4 in mutagen-induced mitotic recombination, although in the wild-type cells it stimulated UV-induced mitotic gene conversion (Vlcˇkova´ et al. 1994). In 1996, it was shown that PSO4 is allelic with PRP19 (Grey et al. 1996), which is an essential gene encoding a spliceosomal complex-associated protein (Cheng et al. 1993). The pleiotropic phenotype of the pso4 mutant suggested that Pso4/Prp19 has more functions and that one of them directly affects the DNA repair complex needed for the function of TLS and recombination (Brendel and Henriques 2001; Revers et al. 2002). It was also speculated that the function of Pso4/Prp19 in DNA repair could be effected either by a direct protein–protein association with the pre-existing recombinosome in a mutagen-treated yeast cell, or via binding to the gaps in DNA resulting from DSB

(Brendel and Henriques 2001; Brendel et al. 2003). Several eukaryotic homologues of Pso4/Prp19 have been identified, indicating evolutionary conservation of the protein. The human homologue of Pso4/Prp19 (HsPso4/ HsPrp19) was found to be a part of the nuclear matrix (Gotzmann et al. 2000; Erdemir et al. 2002; HsPso4/ HsPrp19 was referred to as hNMP 200 in these studies). Consequently, its yeast counterpart could also be a matrix protein and in this way could be involved in various nuclear processes and in genome maintenance, e.g. in the repair and replication of DNA, in transcription and in RNA processing (Gotzmann et al. 2000; Brendel et al. 2003). More recently, HsPso4/HsPrp19 was also identified as a terminal deoxynucleotidyl transferase-associated protein (Mahajan and Mitchell 2003); and this supports the view that HsPso4/HsPrp19 plays an important role in DNA repair processes that result from DSB induction. This is in good accordance with our findings (Morais et al. 1996), which showed that the S. cerevisiae Pso4/Prp19 protein might be involved in a rejoining step during cross-link repair. In 1992, the S. cerevisiae RAD51 gene was cloned and sequenced. Its product (ScRad51) had 54% and 29% overall amino acid sequence similarity and identity, respectively, with the E. coli RecA protein (Shinohara et al. 1992; Aboussekhra et al. 1992; Basile et al. 1992). The most conserved regions of ScRad51 are represented by Walker A and B motifs in the central core region, where RecA and ScRad51 are 61% similar and 35% identical (Bianco et al. 1998). However, there is dissimilarity in the N- and C-terminal parts of the proteins: ScRad51 lacks about 112 amino acids at the C-terminus, but has an extension of about 120 amino acids at the N-terminus, compared with RecA (Shinohara et al. 1992; Basile et al. 1992; Bianco et al. 1998). Nevertheless, ScRad51 was suggested to be a structural and functional homologue of the E. coli RecA protein. The E. coli RecA and S. cerevisiae Rad51 homologues were subsequently identified in a wide range of other eukaryotic organisms, including humans (for a review, see Duda´sˇ and Chovanec 2004). Thus, it was established that the RecA-like proteins comprise a family of highly conserved proteins. The key roles of the Rad51 protein are in the localization of homologous DNA and in DNA strand-exchange reactions; and these are performed by remarkably similar mechanisms in bacteria, yeast and human cells. In contrast to E. coli, where a single RecA protein participates in virtually all HR events, HR in eukaryotic organisms is mediated by a group of genes known as the RAD52 epistasis group. The RAD52 group members in the yeast S. cerevisiae include RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54/TID1, MRE11 and XRS2. Of these, the products of the RAD51 and RAD52 genes are considered to be the pivotal HR proteins. S. cerevisiae rad52 mutants show severe recombination defects and very high sensitivity to DSBinducing agents, such as IR, MMS, bleomycin and 8-MOP + UVA. Although the S. cerevisiae RAD52 gene was cloned and sequenced some time ago, the

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biochemical function and structure of the protein (ScRad52) have only recently been revealed. It was shown that ScRad52 has no structural and functional homology with any prokaryotic recombination protein. Biochemically, ScRad52 non-cooperatively binds both ssDNA and dsDNA, promotes annealing of complementary ssDNA and serves as a mediator protein in the ScRad51-dependent DNA strand-exchange reaction. ScRad52 interacts with ScRad51 and both proteins are components of the HR complex (for a review, see Duda´sˇ and Chovanec 2004). Based on these observations, we speculated that the individual activities of the RecA protein were shared by two different proteins in S. cerevisiae, namely ScRad51 and ScRad52. To verify this, we used the E. coli recA gene as a probe for complementation of the sensitivity of the rad51 and rad52 mutants to MMS, 8-MOP + UVA and UV. In contrast to our first experiments (Cˇerna´kova´ et al. 1991), in which we used the rad52 mutant carrying a point mutation in the RAD52 gene, here we used mutants with inactivating disruptions in the RAD51 and RAD52 genes. As before, the E. coli recA gene was cloned into an extrachromosomally replicating yeast vector downstream of either a constitutive ADH1 promoter to yield pVT103L recA, or an inducible GAL10-CYC1 promoter to produce pYEDP1/8-2recA. Our results showed that RecA was not able to complement the sensitivity of the rad51 strain to MMS and 8-MOP + UVA (Morais et al. 1998; Table 1). This was in line with the report by Shinohara et al. (1993), who showed that the human Rad51 homologue did not complement the DNA repair defects of the S. cerevisiae rad51 mutant. The N-terminal region of ScRad51 was shown to be important for its interaction with ScRad52 (Shinohara et al. 1992; Sung 1994). Sequence alignment analysis revealed that 55 Nterminal amino acid residues of ScRad51 are not present in RecA. We therefore reasoned that the RecA protein could not substitute ScRad51 because it is not able to form an active complex with ScRad52 due to this truncation of the N-terminal region. Alternatively, the different polarity of the DNA strand-transfer reaction mediated by RecA and ScRad51 (Sung and Robberson 1995) may explain why we did not observe RecA-mediated complementation of the rad51 mutant sensitivity. RecA partially increased the resistance of the rad52 mutant cells to MMS, 8-MOP + UVA and IR and fully complemented the DSB repair defect of these cells after their exposure to IR and MMS (Morais et al. 1998; Duda´sˇ et al. 2003; Fig. 2, Table 1). This indicated that ScRad51 might be essential for stimulation of the RecA activity in the rad52 mutant strain. Consistent with this, RecA did not complement the sensitivity of the rad51 rad52 double mutant to 8-MOP + UVA and MMS (Morais et al. 1998). The fact that RecA partially complemented the sensitivity and fully complemented the DSB repair defect of the rad52 mutant cells following exposure to agents inducing DSB indicated that ScRad52, although not homologous to RecA, could possess some RecA-like

activities required for DSB repair. How might the E. coli RecA protein operate in DSB repair in the rad52 mutant cells? One possible explanation was that RecA might be able to replace ScRad52 in the protein–protein interactions performed during DSB repair in yeast (Hays et al. 1995; Krejcˇi et al. 2001). Of the interactions of ScRad52 identified so far, the most important seems to be the interaction with ScRad51 (Hays et al. 1995; Krejcˇi et al. 2001). A physical interaction between ScRad51 and ScRad52 is necessary for ScRad51-mediated DNA strand-invasion and strand-exchange, the two activities that are prerequisites for DSB repair by HR. However, although two-hybrid experiments did not show ScRad51–RecA interaction, they revealed RecA–RecA self-interaction (Duda´sˇ et al. 2003). This suggested that the E. coli RecA protein expressed in yeast is able to form a multimeric complex and hence nucleoprotein filaments, the generation of which represents an early step in HR (Baumann and West 1998). Thus, we prefer the explanation that, in the absence of ScRad52, when there are no endogenous HR events, RecA on its own takes over the processing of strand interruptions in yeast DNA (Duda´sˇ et al. 2003). Taken together, we demonstrated that RecA complemented the defect of the pso4 mutant cells in induced mutagenesis (Fig. 2, Table 1), indicating a similarity of action for RecA and Pso4 in the process. Furthermore, RecA complemented the DSB repair defect of the rad52 mutant cells, although not that of the rad51 mutant cells (Fig. 2, Table 1), suggesting that ScRad52 has some activity, not yet identified, in common with RecA. Our complementation results suggest that ScRad51 must also have this activity. Consistent with this, it was recently shown that ScRad52 has DNA strand-exchange activity (Bi et al. 2004).

Heterologous expression of the nth gene DNA lesions induced by reactive oxygen species (ROS) include base damage, base loss, SSB, DSB and DNA– protein cross-links. The majority of these lesions are primarily repaired via BER (Wallace 1998; Memisoglu and Samson 2000; Brozmanova´ et al. 2001a). BER of altered bases is initiated by DNA glycosylases; and these can be divided into two separate groups, based on the presence or absence of an associated apurinic/apyrimidinic (AP)-lyase activity. In E. coli, endo III (Nth) and endo VIII (Nei) are bifunctional repair enzymes with combined DNA glycosylase and AP-lyase activities. Nth is a DNA repair enzyme, first described as an E. coli endonuclease that makes single-strand incisions in UV-irradiated DNA (Radman 1976). Subsequently, several groups partially purified enzyme activities from E. coli, including X-ray endonuclease (Katcher and Wallace 1983), thymine glycol–DNA glycosylase (Demple and Linn 1980) and urea–DNA glycosylase (Breimer and Lindahl 1984), that were later shown to be functions of Nth. As mentioned, Nth displays both N-glycosylase

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and AP-endonuclease activities; and it has been reported that it is not an endonuclease but rather a lyase catalyzing a b-elimination reaction at the AP sites generated after base removal (Bailly and Verly 1987; Boiteux 1993; Cunningham et al. 1994). It has been suggested that Nglycosylase and the AP-lyase activities are associated in a common reaction pathway (Kow and Wallace 1987; Demple and Harrison 1994). The enzyme recognizes a number of thymine modifications with intact pyrimidine rings, including cis- and trans-thymine glycol (Tg), 5,6dihydro-, 5,6-dihydro-6-hydroxy-, 5,6-dihydrodihydroxy- and 6-hydroxy-5,6-dihydro-thymine and ring fission products such as 5-hydroxy-5-methylhydantoin, urea and formyl-, formylpyruvyl- and methyltartronyl-urea, uracil and cytosine photohydrates and some oxidized guanine residues. These can be generated in DNA by exposure to IR, UV, H2O2, potassium permanganate, osmium tetroxide or sodium bisulfite. The nth gene encoding this enzyme has been cloned and sequenced (Cunningham and Weiss 1985; Asahara et al. 1989; Cunningham et al. 1989). The Nth protein has 221 amino acids and a molecular mass of 23.6 kDa. It is an iron– sulfur protein having a 4Fe–4S cluster. Its crystal structure has also been resolved (Thayer et al. 1995). Although Nei exhibits little if any sequence similarity to Nth (Jiang et al. 1997; Burgess et al. 2002), it has overlapping substrate specificity with Nth for pyrimidine-derived lesions (for reviews, see Wallace 1998; Gros et al. 2002; Dizdaroglu 2003). The E. coli nth mutant is a weak mutator that is not hypersensitive to the lethal effect of H2O2 or IR, suggesting redundancy in the pathways for the repair of oxidized pyrimidine (Cunningham and Weiss 1985). In contrast, the nth nei double mutant is a strong mutator, producing predominantly G:C to A:T transitions, and exhibits increased sensitivity to H2O2 and IR (Jiang et al. 1997; Saito et al. 1997). These observations suggest that endo VIII may serve as a back-up for endo III during DNA repair (Wallace et al. 2003). However, the activity of endo VIII in E. coli is only 10% of the total endonuclease activity and the removal of Tg in E. coli appears to depend heavily on endo III (Melamede et al. 1994). S. cerevisiae possesses two proteins, the functions of which are similar to that of the E. coli Nth protein. These are Ntg1 and Ntg2 (Gossett et al. 1988; Eide et al. 1996; Augeri et al. 1997) and they are composed of 399 and 380 amino acids, respectively. Both Ntg1 and Ntg2 are related to each other (41% identity, 63% similarity) and to E. coli Nth (Girard and Boiteux 1997; You et al. 1998; Bruner et al. 1998; Dizdaroglu 2003). Both proteins contain a highly conserved helix–hairpin–helix motif, although only Ntg2 possesses the iron–sulfur cluster characteristic of Nth and its homologues. Ntg1 is localized to both mitochondria and nucleus, whereas Ntg2 is found only in the nucleus (You et al. 1999). Single and double mutants with disruptions of NTG1 and/or NTG2 display increased mutation frequency after exposure to oxidizing agents (Alseth et al. 1999), although they are not more sensitive to the killing effects

of these agents, even when mutations in NTG1 and NTG2 are combined with deletion of APN1, a product of which is the major AP-endonuclease involved in BER in yeast (Eide et al. 1996; Swanson et al. 1999; Alseth et al. 1999; You et al. 1999; Doetsch et al. 2001). This suggests that an alternative pathway, or a back-up system, operates in S. cerevisiae to handle thymine oxidation products. In attempts to complement a putative defect in the oxidative damage repair pathway of the S. cerevisiae pso3 mutant and to characterize substrates for Nth which might have a role in IR and H2O2 toxicities in wild-type S. cerevisiae cells, we transferred the E. coli nth gene into S. cerevisiae. In order to achieve this, the coding-region of the E. coli nth gene was constructed by site-directed mutagenesis (Sˇkorvaga et al. 2003) and ligated into extrachromosomally replicating yeast expression vectors (see above). We used either the constitutive ADH1 or inducible GAL10–CYC1 promoters to produce pVT103U nth and pADH nth or pYEDP1/82nth recombinant vectors, respectively. The expression of the bacterial Nth protein in cell-free extracts of transformed yeast was in all cases confirmed by Western blotting, using anti-Nth polyclonal antibodies. The S. cerevisiae pso3 mutant (Henriques and Brendel 1990; Henriques et al. 1997; Brendel and Henriques 2001) was shown to have cross-sensitivity to the oxidizing agents H2O2, paraquat and bleomycin, but wildtype resistance to alkylating agents (Brendel et al. 1998; Brozmanova´ et al. 2001b). This strongly suggested a possible function for Pso3 in the repair of oxidative DNA lesions, most probably in the BER pathway. Parallel to several unsuccessful attempts to clone the PSO3 gene, it was reasonable to further analyze the putative repair defects of the S. cerevisiae pso3 cells. We showed (Brozmanova´ et al. 2001b) that the basis of the sensitivity of the pso3 mutant cells to H2O2 is their increased ability to undergo DNA double-strand breakage induced by this agent. We found that expression of the nth gene in the pso3 mutant strains increased survival, decreased mutability and protected yeast genomic DNA from breakage following H2O2 treatment (Fig. 3, Table 1). Therefore, we concluded that Pso3 might be a DNA repair protein with some functional similarity to the E. coli Nth protein. However, the molecular role of Pso3 in the repair of oxidative DNA damage in yeast remains to be elucidated. Subsequent results, in which either BER or HR were inactivated in the pso3 background by disruption of NTG1 and/or NTG2, or RAD51, suggested a role of the PSO3 gene product in the oxidative repair pathway distinct from BER (Duda´sˇ et al., unpublished data). Approximately 70% of IR-induced DNA damage is formed by ROS produced by radiolysis of water in the vicinity of the DNA molecule (Ward 1985). Although H2O2 produces a spectrum of hydroxyl radical-mediated base damage that overlaps with that produced by IR, the spectrum of primary DNA lesions induced by these agents is different. It was proposed that lesions induced by IR are

326 Fig. 3 Results obtained by heterologous expression of the E. coli nth gene in the yeast S. cerevisiae. Only results that gave either positive or negative results are presented

more cytotoxic than those induced by H2O2 due to their spatial distribution, which affects their capacity to be repaired (Ward 1981). We examined the contribution of Nth-repairable lesions to the cytotoxic effects of IR and H2O2 in S. cerevisiae and found (Sˇkorvaga et al. 2003) that pADH nth-transformed wild-type yeast were more resistant to the toxic effect of IR than control pADH transformants (Fig. 3, Table 1). These results indicated that DNA damage-types that are substrates for Nth, i.e. modified thymine residues and/or apurinic sites, might represent potentially lethal lesions in yeast; and hence radiation survival is determined to a large extent by the efficiency of the endogenous repair system(s) for such lesions. However, our results showed that Nth did not confer resistance to H2O2 in the individual wild-type strains of different origin (Brozmanova´ et al. 2001b; Sˇkorvaga et al. 2003). It therefore seems either that the H2O2-sensitivity phenotype is not solely related to Nthrepairable lesions in yeast or, more likely, that there can be alternative DNA repair activities for such lesions in this organism. No effect of nth expression on sensitivity to IR and H2O2 was found in rad52 mutant cells (Table 1) and this is similar to the requirement of the Rad52 protein for the functioning of the E. coli ada-encoded MTase in yeast. These results suggest that heterologously expressed bacterial DNA repair proteins might not function in rad52 mutant cells as effectively as in wild-type cells. In summary, our results using nth confirmed the suggestion that Pso3 is a DNA repair protein with some degree of functional similarity to the E. coli Nth protein. Pso3 is presumably involved in cellular response to oxidative damage through its ability to prevent DNA double-strand breakage. Furthermore, we provided evidence that DNA lesions that are substrates for Nth can also make a contribution to the toxic effect of IR in wild-type yeast. Hence, DSB should not be considered the sole lethal lesions following IR exposure.

Concluding remarks Heterologous expression experiments have been highly successful for a wide range of genes, particularly those

encoding DNA repair factors. These experiments led to: (1) exploring the biological and biochemical features of numerous DNA repair enzymes and proteins, (2) addressing the biological relevance of the biochemical differences observed among DNA repair enzymes and proteins from different organisms, (3) identifying new DNA repair genes due to their powerful cloning strategy and (4) providing host cells with additional resistance to the corresponding type of DNA lesion (for a review, see Memisoglu and Samson 1996). The E. coli genes ada (encoding a protein involved in the repair of alkylation damage to DNA), recA (encoding the main recombinase in E. coli) and nth (a product of which is responsible for the repair of oxidative base damage to DNA) were heterologously expressed in the budding yeast S. cerevisiae (Brozmanova´ et al. 1990, 1991, 1994, 2001b; Cˇerna´kova´ et al. 1991; Morais et al. 1994, 1996, 1998; Vlcˇkova´ et al. 1994, 1997; Slaninova´ et al. 1996; Farkasˇ ova´ et al. 2000; Duda´sˇ et al. 2003; Sˇkorvaga et al. 2003; Table 1) and in other systems (Kido et al. 1992; Reiss et al. 1996, 1997, 2000; Shcherbakova et al. 2000; see references cited in Memisoglu and Samson 1996). These studies unequivocally showed that the encoded DNA repair factors can function efficiently in heterologous cells, suggesting that the DNA lesions they recognize are structurally identical, irrespective of genome organization. The aim of our heterologous expression experiments was to increase the capacity of S. cerevisiae wild-type cells to deal with Ada-, RecA- and Nth-repairable DNA lesions and to complement the repair defects of the particular mutants (see text above). Using our approach, we provided evidence that the demethylation repair pathway per se can act on yeast DNA, thereby indirectly suggesting that a similar endogenous mechanism exists in S. cerevisiae cells (Brozmanova´ et al. 1990). Consistent with this, cloning of the S. cerevisiae MTase gene, MGT1, was reported 1 year later (Xiao et al. 1991) and the in vivo role of its product was subsequently determined (Xiao and Samson 1992). Moreover, we showed that the total amount of MTase in a cell does not necessarily correlate with the protection provided (Farkasˇ ova´ et al. 2000) and that there may be a possible co-factor requirement for MTase in eukaryotic cells (Brozmanova´ et al. 1994). These

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two facts may have important implications in the potential use of MTase genes and cDNAs in gene therapy. Regarding heterologous expression experiments using the E. coli recA gene, we suggested that the S. cerevisiae Rad52 protein has some activity, not identified up to that time, in common with RecA/Rad51 (Duda´sˇ et al. 2003). Indeed, a new activity was recently reported for ScRad52, which was shown to carry out a DNA strand-exchange reaction (Bi et al. 2004). In addition, we reported some degree of functional similarity between RecA and Pso4 in induced mutagenesis (Morais et al. 1994; Slaninova´ et al. 1996) and between Nth and Pso3 in the repair of oxidative DNA damage (Brozmanova´ et al. 2001b). Finally, we provided evidence that DNA lesions that are substrates for Nth can also make a contribution to the toxic effects of IR in wild-type yeast and that, therefore, DSB should not be considered the sole lethal lesions following IR exposure. Taken together, we showed that heterologous expression of DNA repair enzymes and proteins from E. coli was successfully used to address some questions about DNA repair processes in S. cerevisiae cells. Acknowledgements The authors thank Dr. G.P. Margison and Dr. Z. Duda´sˇ ova´ for critical reading of the manuscript. Work in the authors’ laboratory is supported by the VEGA Grant Agency of the Slovak Republic (grants 2/3091/23, 1/0043/03) and by project 2003 SP 51 028 08 00/028 08 01 in the national program Use of Cancer Genomics to Improve the Human Population Health.

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