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Nucleic Acids Research Pages 3077-3083  

Saccharomyces cerevisiae exonuclease-1 plays a role in UV resistance that is distinct from nucleotide excision repair
Introduction
Materials And Methods
   Materials
   Sequence alignment
   Northern blotting analysis
   Gene disruption
   DNA damage treatment
Results
   Two S.cerevisiae homologs of S.pombe Exo1
   Expression of EXO1 is UV inducible
   The role of EXO1 and DIN7 in DNA repair
   Epstasis analysis
Discussion
Acknowledgements
References

Saccharomyces cerevisiae exonuclease-1 plays a role in UV resistance that is distinct from nucleotide excision repair

Saccharomyces cerevisiae exonuclease-1 plays a role in UV resistance that is distinct from nucleotide excision repair

Junzhuan Qiu, Min-Xin Guan2, Adam M. Bailis1, Binghui Shen*

Department of Cell and Tumor Biology and 1Department of Molecular Biology, The Beckman Research Institute of City of Hope, Duarte, CA 91010-3000, USA and 2Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA

Received April 17, 1998; Revised and Accepted May 11, 1998

ABSTRACT

Two closely related genes, EXO1 and DIN7, in the budding yeast Saccharomyces cerevisiae have been found to be sequence homologs of the exo1 gene from the fission yeast Schizosaccharomyces pombe. The proteins encoded by these genes belong to the Rad2/XPG and Rad27/FEN-1 families, which are structure-specific nucleases functioning in DNA repair. An XPG nuclease deficiency in humans is one cause of xeroderma pigmentosum and those afflicted display a hypersensitivity to UV light. Deletion of the RAD2 gene in S.cerevisiae also causes UV hypersensitivity, due to a defect in nucleotide excision repair (NER), but residual UV resistance remains. In this report, we describe evidence for the residual repair of UV damage to DNA that is dependent upon Exo1 nuclease. Expression of the EXO1 gene is UV inducible. Genetic analysis indicates that the EXO1 gene is involved in a NER-independent pathway for UV repair, as exo1 rad2 double mutants are more sensitive to UV than either the rad2 or exo1 single mutants. Since the roles of EXO1 in mismatch repair and recombination have been established, double mutants were constructed to examine the possible relationship between the role of EXO1 in UV resistance and its roles in other pathways for repair of UV damaged DNA. The exo1 msh2, exo1 rad51, rad2 rad51 and rad2 msh2 double mutants were all more sensitive to UV than their respective pairs of single mutants. This suggests that the observed UV sensitivity of the exo1 deletion mutant is unlikely to be due to its functional deficiencies in MMR, recombination or NER. Further, it suggests that the EXO1, RAD51 and MSH2 genes control independent mechanisms for the maintenance of UV resistance.

INTRODUCTION

Multiple DNA repair pathways exist to counteract the deleterious effects of UV irradiation on cellular DNA. One of the widely distributed repair enzymes, photolyase, which utilizes visible light to reverse UV-induced DNA damage, is conserved from prokaryotic to eukaryotic cells (1). Another important and conserved pathway for the repair of UV damage to DNA is nucleotide excision repair (NER). This pathway works by incising DNA on either side of the damage site with endonucleases, removing a 29 bp oligonucleotide with a helicase and then resealing the gap with polymerase and ligase. Seventeen polypeptides are required in this process in mammalian cells (2). Defects in the polypeptides that execute the early stages of recognition and cleavage cause human genetic diseases, including xeroderma pigmentosum, which predisposes patients to skin cancer (2-4).

NER is a mechanism by which cells can excise UV photoproducts, such as cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs) from DNA. In Saccharomyces cerevisiae, mutation of the genes that encode key NER enzymes causes dramatic UV sensitivity (5-7). However, in the fission yeast Schizosaccharomyces pombe deletion of the NER genes confers less sensitivity to UV, such that the mutants retain a substantial ability to remove UV photoproducts (8,9). This implies the existence of a secondary pathway for UV damage repair in S.pombe and an enzyme (SPDE) that initiates this process has been discovered and preliminarily characterized (10,11).

Recently, another nuclease, exonuclease-1 (SpExo1) has been isolated from S.pombe (12,13). The enzyme activity and mRNA expression levels are significantly induced during meiosis, indicative of its function in mismatch correction during meiotic recombination. Exo1 is a member of a family of DNA repair nucleases that includes Rad13 and Rad2 from S.pombe and their human homologs XPG and FEN-1 (13). Rad13 is a key component of the NER machinery in S.pombe, acting as a 3[prime] nuclease during pyrimidine dimer removal. Accordingly, rad13 null mutants are sensitive to UV irradiation (14). The null mutant of rad2 is modestly sensitive to UV, implying that it plays a minor role in UV repair (15).

The RAD2 and RAD27 genes from S.cerevisiae are homologs of the S.pombe Rad13 and Rad2 genes respectively. Deletion of both the RAD2 and RAD27 genes has a synergistic effect on repair of UV-induced DNA damage in S.cerevisiae (16). This result indicates that there is more than one pathway for UV resistance in S.cerevisiae as well. Moreover, residual UV survival exists even in the rad2 rad27 double mutant, implying the existence of additional pathways for UV resistance, such as recombinational repair and DNA replication bypass. Therefore, we propose that an additional Rad2-like nuclease may play a role in one of these pathways for UV resistance. One candidate is an exonuclease purified from S.cerevisiae that is homologous to S.pombe Exo1, which has been named ScExo1 (17). In order to study the role of S.cerevisiae homologs of S.pombe Exo1 in DNA repair, we have cloned two genes from S.cerevisiae that are substantially homologous to S.pombe Exo1. Here we report the genetic consequences of deleting these two genes in S.cerevisiae and their functional relationship to NER and other UV resistance mechanisms.


Table 1. Primers used to construct and confirm mutants

* The long primer includes a DNA sequence encoding six histidines (Histag), which can be used to purify the enzyme after it is overexpressed. Underlined sequences were designed for the amplification of HIS3 mark, the remaining sequences of KNSCB1 and KNSCB2 are from DIN7.


Table 2. Saccharomyces cerevisiae strains used in the study

aAll the strains are derivatives of W1021-7c and W1089-6c. Differences in the genotype from the parental strains are indicated in the table.

MATERIALS AND METHODS

Materials

Oligonucleotide primers synthesized in the City of Hope Cancer Center core facility and used for amplifying genes and constructing null mutant strains are listed in Table 1. The yeast strains used in this study are listed in Table . The vector pET-28b was from Novagen (Madison, WI) and Escherichia coli strain XL2 blue and pBSK vector were from Stratagene (La Jolla, CA). Restriction enzymes were obtained from New England Biolabs (Beverly, MA). [[alpha]-32P]dCTP was purchased from NEN (Boston, MA). Yeast culture media, including YPD, synthetic complete (SC), minimal sporulation and synthetic dextrose minimal (SD), were prepared according to Sherman et al. (18). Amino acids and all other medium components and chemicals were purchased from Sigma (St Louis, MO).

Sequence alignment

Sequences of the relevant genes were obtained from the NCBI protein database. They were then compared using Optimal Global Alignment of Two Sequences at EERIE (Nimes, France). The percentages of identical and similar amino acids were calculated.

Northern blotting analysis

A wild-type yeast strain W1021-7c was grown in 50 ml YPD to late log phase. Cells were harvested and resuspended in 10 ml water. The resuspended cells were spread on two sets of YPD agar plates. One set of the plates was irradiated with 80 J/m2 while the other set was kept as a control. Following irradiation, cells were collected from the plates, 2 ml aliquots made into four cultures, spun down, resuspended in 10 ml YPD liquid and cultured at 30°C. Cells were harvested at the indicated times after irradiation and their total RNA was isolated using RNeasy Mini Kits (Qiagen, Santa Clarita, CA). An aliquot of 10 µg total RNA was fractionated by electrophoresis in a 1.4% agarose-formaldehyde gel, transferred to a Zeta-probe membrane (BioRad) and hybridized with EXO1 and ACT1, [32P]dCTP-labeled by random priming (19). The images were produced by exposing the filters to X-ray film (Kodak). Specific EXO1 mRNA bands in Figure 2A were scanned and quantitated using the IPLabGel system (Stratagene) running MacBAS software.

Gene disruption

For construction of the exo1 null mutant strain, the EXO1 gene was amplified by PCR using primers with HindIII and NcoI sites (exo1p1 and exo1p2, Table 1) and cloned into the vector pET-28b (Novagen). The URA3 gene was obtained from plasmid YEp24 and inserted into two PstI sites, replacing the EXO1 coding sequence between positions 170 and 828. The fragment containing the exo1::URA3 disruption was removed from the plasmid with XbaI and NotI and transformed into yeast strains W1021-7c and W10896c (see Table 2), selecting for cells that could grow without uracil. Ura+ transformants were analyzed by PCR using primers exo1p3 and exo1p4 (Table 1) and genomic Southern blotting to verify disruption of EXO1.

The DIN7 gene was amplified by PCR using a pair of primers (din7p1 and din7p2, Table 1) with restriction sites NcoI and BamHI and cloned into pBSK vector (Stratagene). The gene was disrupted by transforming with a DNA fragment containing the HIS3 gene flanked by two 53 bp fragments homologous to nt 25-78 and 1273-1326 of the DIN7 coding sequence (KNSCB1 and KNSCB2 in Table 1). The HIS3 coding sequence was amplified from a genomic clone, HIS3MX, kindly provided by Dr K.Kuchler. His+ transformants were checked for disruption of DIN7 using PCR (primers din7p3 and din7p4 in Table 1). Double null mutants were obtained using standard genetic techniques for manipulation of S.cerevisiae (18).

A msh2 mutant was generated with strain FDAB13A using plasmid pEAI98, which contains the hisG-URA3-hisG universal disrupter flanked by 350 bp of DNA upstream of MSH2 and 1.2 kb of DNA from the 3[prime]-end of the MSH2 open reading frame. After digestion of pEAI 98 with AatII and PvuII, the msh2::hisG::URA3::hisG fragment was gel purified and transformed into FDAB13A cells. Ura+ transformants were selected on minimal medium without uracil and analyzed by PCR for disruption of MSH2. The msh2 single mutant transformants were crossed with a rad2 single mutant, 135-2B, to obtain rad2 msh2 double mutants using standard genetic techniques (18).

The rad2 rad51 double mutant was obtained by crossing rad2 (135-2A) and rad51 (U687) single mutants. The exo1 rad51 and exo1 msh2 double mutants were obtained by crossing exo1 (FDAB15D) with rad51 (U687) or msh2 (FDABFT) mutants individually. All disruption mutants established in this study are listed in Table 2.

DNA damage treatment

Strains were grown in YPD liquid to saturation at 30°C and cell density was measured with a spectrophotometer (600 nm), diluted in water and plated on YPD agar. The plates were exposed to different UV dosages (254 nm germicidal lamp) and incubated in the dark for 3-4 days at 30°C before colonies were counted. Survival rate was determined based on the ratio between colony counts with and without UV treatment. Survival curves represent the average from at least two independent experiments with two sets of isogenic strains.

To measure [gamma]-ray survival, cells were grown to stationary phase in YPD at 30°C, washed, diluted and aliquots put into Petri dishes for irradiation. After irradiation, various dilutions were plated on YPD agar plates and incubated at 30°C for 4 days. The method of MMS treatment followed Reagan et al. (16), where cells are grown to stationary phase at 30°C, harvested, washed with water and resuspended in 0.1 M potassium phosphate buffer (pH 7). After treatment with MMS (0, 0.1, 0.2 and 0.3%) for 30 min at 30°C with shaking, the cells were washed four times with water, diluted and plated. The plates were incubated at 30°C for 3-4 days before counting. Calculation of survival rate for the [gamma]-ray and MMS treatments was the same as that for UV treatment.

RESULTS

Two S.cerevisiae homologs of S.pombe Exo1

We searched the publicly available S.cerevisiae sequence databases (GenBank/EMBL/NCBL) using the S.pombe Exo1 protein sequence as a query in the BlastP search algorithm (21). This search led to the identification of two open reading frames encoding proteins with a high degree of homology to SpExo1. They are ScEXO1 and DIN7. ScEXO1 on chromosome XI (YOR033C, accession no. Z74941) is predicted to encode a 702 amino acid protein, while DIN7 (22) on chromosome IV (YD93208B02C, accession no. Z70202) encodes a 430 amino acid protein. Using primers designed from these sequences (exop1 and exop2 for the EXO1 gene and din7p1 and din7p2 for the DIN7 gene, Table 1), both genes were obtained by PCR and cloned into plasmid vectors pET28b and pBSK respectively and sequenced. The nucleotide sequence of the coding regions of these two genes are identical to the sequences deposited in the databases.

Comparison of the predicted polypeptide sequences for ScExo1, ScDin7 and SpExo1 revealed a conserved nuclease domain at their N-termini, from amino acid residue 1 to 370. The amino acid sequence identity between SpExo1 and ScExo1 and ScDin7 is ~35%, while the identity between the ScExo1 and Din7 is 56%. The interspecific sequence similarity is ~65%, while the intraspecific similarity is ~85%. The protein sequence comparison for this exonuclease subfamily is depicted in Figure 1. From these data, both of the proteins appear to be structural homologs of S.pombe exonuclease-1.


Figure 1. Sequence comparison of S.cerevisiae Exo1 and Din7 and S.pombe Exo1 proteins. (A) The regions of proteins compared in this study. (B) Alignment of the N-terminal amino acid sequences. The alignment was created using Optimal Global Alignment of Two Sequences at EERIE (Nimes, France). The identical amino acid residues are indicated by a vertical line while similar amino acids are indicated by a colon; (C), the percentage of identical and similar amino acid residues among N-termini of the proteins.
Figure 2. Gene expression in response to UV treatment. (A) Total RNA was prepared from the wild-type strain W1021-7c (Table 1) harvested at the time indicated after the UV treatment. For each time point, 10 µg total RNA were fractionated by electrophoresis in a 1.4% agarose-formaldehyde gel, transferred to a Zeta-probe membrane (BioRad) and hybridized with probes of ScEXO1 encoding exonuclease-1 and ACT1 for the yeast actin gene. Both of the DNA fragments were [32P]dCTP-labeled by random priming (19). (B) Intensities of specific mRNA bands in (A) were scanned and quantitated using the IPLabGel system (Strategene) running MacBAS software.


Figure 3. UV survival of wild-type, [Delta]exo1, [Delta]din7 and [Delta]exo1[Delta]din7 strains of S.cerevisiae. Isogenic strains FDAB15B, FDAB16C (WT), FDAB15D, FDAB16D ([Delta]exo1), FDAB15C, FDAB16B ([Delta]din7) and FDAB15A, FDAB16A ([Delta]exo1[Delta]din7) were grown at 30°C and cells were plated and UV irradiated, followed by incubation of the plates at 30°C in the dark. Similar results were obtained with wild-type strain W1089-6c and isogenic single mutants. The data were averaged from at least three independent experiments with two sets of isogenic strains.

Expression of EXO1 is UV inducible

Previously, it was found that expression of DIN7, like the NER gene RAD2 (23,24), is UV inducible in S.cerevisiae (22). In order to determine the UV inducibility of the EXO1 gene, EXO1 mRNA levels were assessed both prior to and following exposure to UV. As shown in Figure 2A, EXO1 gene expression is UV inducible. The specific mRNA levels increased 7-fold by 5 h after UV exposure, while the EXO1 expression levels in cells grown under the same conditions without UV treatment remained unchanged (Fig. 2B).

The role of EXO1 and DIN7 in DNA repair

In order to test whether EXO1 and DIN7 function in DNA repair, we have constructed null mutants (Table 2). Both null mutants are viable, as is the exo1 din7 double mutant obtained from crosses. All three strains grew at the wild-type rate at both 30 and 37°C. Sensitivity to [gamma]-ray and UV irradiation and to MMS was assessed in the single and double mutants. Neither the single mutants nor the double mutant displayed any sensitivity to [gamma]-rays or MMS (data not shown). However, the exo1 null mutant displayed a mild sensitivity to UV, indicating that it plays a minor role in repair of UV damage to DNA. In contrast, the din7 mutant exhibited no sensitivity to UV, indicating that it does not play a role in UV survival. Consistent with these observations, the exo1 din7 double mutant displayed the same UV sensitivity as the exo1 single mutant (Fig. 3).

Epstasis analysis

The UV sensitivity of the exo1 mutant suggests that EXO1 plays a role in UV repair. As discussed previously, EXO1 could encode a component of an alternative mechanism for UV repair that is distinct from NER. To test this, we crossed the exo1 deletion mutant with a rad2 null NER defective strain and analyzed UV survival of rad2 exo1 double mutant segregants (Table 2 and Fig. 4). The results show that the double mutant is more sensitive than either the rad2 or exo1 single mutants, suggesting that the EXO1 gene encodes a component of an NER-independent UV repair pathway in S.cerevisiae.


Figure 4. Epistasis analysis of the exo1::URA3 mutation. Double deletion mutant strains FDAR2-3B and FDAR2-5D were created by crossing the single mutant strains FDAB16D ([Delta]exo1) and 135-2B ([Delta]rad2). The experimental procedure is as described in Figure 3 except lower UV dosages were used. The data were averaged from at least three independent experiments with two sets of isogenic strains.

Because it is known that Exo1 participates in double-strand break-induced recombination (17) and also interacts with the mismatch repair protein Msh2 (25), it is possible that the UV sensitivity of the exo1 mutant is due to a defect in double-strand break repair (DSBR) and/or mismatch repair (MMR). In order to test this possibility, we constructed double mutants of exo1 with msh2 and rad51, two major components of the MMR and DSBR pathways in S.cerevisiae (Table 2). When the mutants were treated with high UV dosages, the msh2 single mutant displayed the same UV sensitivity as the exo1 mutant, while the rad51 deletion mutant was more UV sensitive. Interestingly, the exo1 msh2 and exo1 rad51 double mutants exhibited responses to UV light that were stronger than the corresponding single mutants (Fig. 5). This synergism suggests that the UV sensitivity of the exo1 deletion mutant may not be due to deficiencies in MMR or DSBR.

To further investigate the relationship between the different DNA repair mutations with respect to UV resistance, we have extended the epistasis analysis to include the rad2 mutation. Our results indicate that the rad2, exo1, rad51 and msh2 genes participate in distinct mechanisms for UV resistance because all possible double mutant combinations are more sensitive than the corresponding single mutants (Table 3).

DISCUSSION

ScExo1, ScDin7 and SpExo1 belong to a family of DNA repair enzymes with structure-specific nuclease activity (24,26). Based on sequence comparisons and their biochemical and biological functions in different pathways, these enzymes can be divided into three subfamilies: one includes S.cerevisiae Rad2 and its S.pombe and human homologs Rad13 and XPG; another consists of ScRad27, SpRad2 and hFEN-1; exonuclease-1 belongs to the third. These evolutionarily conserved enzymes may possess complementary biological functions. For example, the ScRad2 subfamily plays a major role in NER. In addition to its role in DNA replication, Rad27 also plays a minor role in UV resistance (14,16,26-29). This led us to study the role of ScExo1 and ScDin7 in UV-induced DNA damage repair. Indeed, our results show that both EXO1 and DIN7 are UV inducible and deletion of EXO1 confers a mild UV sensitivity similar to UVDE deletion mutants in S.pombe (10,30,31; see also below). Furthermore, the epistasis analysis suggests that EXO1 does not play a role in the NER pathway, but instead is involved in a distinct mechanism of UV resistance.


Figure 5. Synergistic effects of the exo1 with rad51 and msh2 mutations. The exo1 rad51 and exo1 msh2 double mutants were made by crossing the individual single mutants and treating with relatively high dosages to study the synergistic effects of these double mutants. The data was averaged from at least three independent experiments with two sets of isogenic strains.

Although Exo1 and Din7 are similar in their N-terminal protein sequences and UV inducibility, it is clear that DIN7 does not play an essential role in UV damage repair. One possibility is that Din7 participates in other DNA repair pathways that do not contribute directly to UV survival. Alternatively, it may play a role in UV damage repair but its loss does not affect survival rate due to redundancy of similar components.

The results shown in Figure 2A and B clearly demonstrate that EXO1 expression is UV inducible. However, these data did not indicate if the damage inducibility of EXO1 is specific for UV light, even though deletion of EXO1 leads only to UV sensitivity and not sensitivity to [gamma]-ray or MMS treatments. Because we found that the distribution of cells at various points in the cell cycle does not change dramatically following UV treatment (our unpublished observation), we also do not believe that inducibility of the EXO1 gene is a result of cell cycle regulation.

Recently, functional analysis of the Exo1 enzyme has been carried out in several laboratories (13,17,25). Exonuclease-1 activity has been demonstrated to be highly inducible during meiosis in S.pombe and is involved in the late steps of homologous recombination, i.e. correction of mismatched base pairs resulting from hybrid DNA formation (13). It has also been strongly indicated by in vitro and in vivo experiments that S.cerevisiae Exo1 functions in mitotic recombination (17). ScExo1 also interacts with Msh2, a major recognition protein in the DNA mismatch repair pathway (32). Since Msh2 is also involved in recombination, it is not clear whether the Exo1-Msh2 complex identified is involved in MMR, recombination or both. However, it is reasonable to assume that the major function of ScExo1 nuclease is in MMR and recombination.

Our observation that ScExo1 functions in UV damage repair via a distinct pathway from NER raises an interesting question: is the observed effect of the exo1 deletion mutation on UV resistance derived from the role of EXO1 in recombination and/or MMR? In order to determine if there is a relationship between the UV resistance function of EXO1 and its roles in recombination and MMR, an epistasis analysis with mutations that cause defects in recombination and MMR was employed. Our results indicate that while a recombination-defective rad51 mutant and a MMR-deficient msh2 mutant both display minor UV sensitivity, the exo1 rad51 and exo1 msh2 double mutants were both more UV sensitive than the corresponding single mutants. This suggests that the UV resistance mediated by EXO1 may be separate from its roles in recombination and MMR. Interestingly, the rad2 rad51 and rad2 msh2 mutants were both more UV sensitive than the corresponding single mutants, indicating that the RAD2, RAD51, MSH2 and EXO1 genes all make distinct contributions to the UV resistance of S.cerevisiae. We speculate that this could represent the repair of distinct classes of DNA lesions or could represent distinct UV damage bypass mechanisms.

Table 3. UV survival of wild-type and mutant S.cerevisiae strains
UV dose Strains (percent survival)a
(J/m2) wt exo1 msh2 rad51 rad2 rad2exo1 rad2rad51 rad2msh2
0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
1 nd nd nd nd 94.0 81.0 45.0 69.0
3 nd nd nd nd 42.0 22.0 6.0 29.0
4 nd nd nd nd 19.0 6.0 1.0 7.0
5 99.5 99.4 99.6 99.4 3.5 0.8 0.1 1.0
aIsogenic wild-type and mutant strains were grown to stationary phase and appropriate dilutions plated onto YPD agar prior to irradiaton and incubation at 30°C for 3-5 days in the dark. Percent survival was calculated as the number of cells surviving to form a colony after UV exposure divided by the number of irradiated cells (0 J/m2) that could form colonies. nd, survival was not determined.

If the UV resistance function of Exo1 nuclease is not related to its roles in DNA recombination and MMR, what other possibilities should be considered? In the fission yeast S.pombe, an NER-independent UV damage repair pathway has been well defined. This pathway possesses a considerable capacity to remove CPDs and 6-4 PPs by a mechanism that is distinct from NER or base excision repair (BER) (8,9,34). The enzyme, named S.pombe DNA endonuclease (SPDE) or UV damage endonuclease (UVDE), has been isolated and characterized (11,35). Meanwhile, the SpRad2 nuclease has been proposed to be the second component of this pathway, acting as a 5[prime] flap endonuclease in removal of the adducted DNA fragment which was released by SPDE (36). The fact that similar synergistic effects are observed in both S.cerevisiae rad2 exo1 and S.pombe rad2 UVDE double mutants (10,30) supports the notion that S.cerevisiae possesses a minor NER-independent pathway as well. However, it was indicated that photoproducts were not removed from DNA in S.cerevisiae NER mutants using antibodies directed against CPD and 6-4 PPs (37). Therefore, we must consider the possibility that residual UV resistance is the result of other mechanisms, such as error-prone repair, that permit DNA to bypass or shuffle damaged bases rather than excise them. Exo1 has been suggested to participate in RNA primer removal during lagging strand DNA synthesis in DNA replication (26; our unpublished data). It may play a role in the bypass of UV lesions by maintaining DNA replication in abnormal circumstances, such as after a high dosage of UV.

This work presents evidence that ScExo1 plays a role in UV resistance in addition to its roles in DNA recombination and mismatch repair (17,25). The information made available in this report will be useful in further functional analysis of the newly emerging family of nucleases in DNA replication, repair and recombination.

ACKNOWLEDGEMENTS

We thank Karl Kuchler (University and Biocenter of Vienna, Vienna, Austria) for additional technical suggestions on the genetic manipulation of yeast. We also thank Rod Rothstein (Columbia University, New York, NY) for yeast strains. We thank Richard Kolodner (Ludwig Cancer Institute, San Diego, CA) and Eric Alani (Cornell University, Ithaca, NY) for plasmid pEAI98. B.H.S. and A.M.B. are members of the NCI designated clinical cancer research center at the City of Hope. This research was supported by City of Hope institutional funds to A.M.B. and B.H.S. and a NIH/NCI grant to B.H.S. (CA73764-01).

REFERENCES

1. Friedberg,E.C., Walker,G.C. and Siede,W. (1995) DNA Repair and Mutagenesis. ASM Press, Washington, DC.

2. Sancar,A. (1994) Science, 22, 1954-1956.

3. Scherly,D., Nouspikel,T., Coriet,J., Ucla,C., Bairoch,A. and Clarkson,S.G. (1993) Nature, 363, 182-184. MEDLINE Abstract

4. O'Donovan,A. and Wood,R.D. (1993) Nature, 363, 185-188. MEDLINE Abstract

5. Unrau,P., Wheatcroft,R. and Cox,B.S. (1971) Biochim. Biophys. Acta, 269, 311-321.

6. Prakash,L. (1975) J. Mol. Biol., 98, 781-795. MEDLINE Abstract

7. Prakash,L. (1977) Mutat. Res., 45, 13-20. MEDLINE Abstract

8. McCready,S., Carr,A.M. and Lehman,A.R. (1993) Mol. Microbiol., 10, 885-890. MEDLINE Abstract

9. McCready,S. and Cox,B. (1993) Mutat. Res., 293, 233-240. MEDLINE Abstract

10. Freyer,G.A., Davey,S. Ferrer,J.V., Martin,A.M., Beach,D. and Doetsch,P.W. (1995) Mol. Cell. Biol., 15, 4572-4577. MEDLINE Abstract

11. Bowman,K.K., Sidik,K., Smith,C.A., Taylor,J.S., Doetsch,P.W. and Freyer,G.A. (1994) Nucleic Acids Res., 22, 3026-3032. MEDLINE Abstract

12. Szankasi,P. and Smith,G.R. (1992) J. Biol. Chem., 267, 3014-3023. MEDLINE Abstract

13. Szankasi,P. and Smith,G.R. (1995) Science, 267, 1166-1168. MEDLINE Abstract

14. Carr,A.M., Sheldrick,K.S., Murray,J.M., Al-Harithy,R., Watts,F.Z. and Lehmann,A.R. (1993) Nucleic Acids Res., 21, 1345-1349. MEDLINE Abstract

15. Murray,J.M., Tavassoli,M., Al-Harithy,R., Sheldrick,K.S., Lehmann,A.R., Carr,A.M. and Watts,F.Z. (1994) Mol. Cell. Biol., 14, 4878-4888. MEDLINE Abstract

16. Reagan,M.S., Pittenger,C., Siede,W. and Friedberg,E.C. (1995) J. Bacteriol., 177, 364-371. MEDLINE Abstract

17. Fiorentini,P., Huang,K.N., Tishkoff,D.X., Kolodner,R.D. and Symington,L.S. (1997) Mol. Cell. Biol., 17, 2764-2773. MEDLINE Abstract

18. Sherman,F., Fink,G.R. and Hicks,J.B. (1986) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Habor, NY.

19. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press,Cold Spring Habor, NY.

20. Gangloff,S., McDonald,J.P., Bendixen,C., Arthur,L. and Rothstein,R. (1994) Mol. Cell. Biol., 14, 8391-8398. MEDLINE Abstract

21. Gish,W. and States,D.J. (1993) Nature Genet., 3, 266-372. MEDLINE Abstract

22. Mieczkowski,P.A., Fikus,M.U. and Ciesla,Z. (1997) Mol. Gen. Genet., 253, 655-665. MEDLINE Abstract

23. Robinson,G., Nicolet,C., Kalainov,D. and Friedberg,E.C. (1986)Proc. Natl. Acad. Sci. USA, 83, 1842-1846. MEDLINE Abstract

24. Lieber,M. (1997) BioEssays, 19, 233-240 MEDLINE Abstract

25. Tishkoff,D.X., Boerger,A.L., Bertrand,P., Filosi,N., Gaida,G.M., Kane,M.F. and Kolodner,R.D. (1997) Proc. Natl. Acad. Sci. USA, 94, 7487-7492.

26. Bambara,R.A., Murante,R.S. and Henricksen,L.A. (1997) J. Biol. Chem., 272, 4647-4650. MEDLINE Abstract

27. Madura,K. and Prakash,S. (1986) J. Bacteriol., 166, 914-923. MEDLINE Abstract

28. Johnson,R.E., Kovvali,G.K., Prakash,L. and Prakash,S. (1995) Science, 269, 238-240. MEDLINE Abstract

29. Tishkoff,D.X., Filosi,N., Gaida,G.M. and Kolodner,R.D. (1997) Cell, 88, 253-263. MEDLINE Abstract

30. Yonemasu,R., McCready,S.J., Murray,J.M., Osman,F., Takao,M., Yamamoto,K., Lehmann,A.R. and Yasui,A. (1997) Nucleic Acids Res., 25, 1553-1558. MEDLINE Abstract

31. Davey,S., Nass,M.L., Ferrer,J.V., Sidik,K., Eisenberger,A., Mitchell,D.L. and Freyer,G.A. (1997) Nucleic Acids Res., 25, 1002-1008. MEDLINE Abstract

32. Thomas,B.J. and Rothstein,R. (1989) Genetics, 123, 725-738. MEDLINE Abstract

33. Sidik,K., Lieberman,H.B. and Freyer,G.A. (1992) Proc. Natl. Acad. Sci. USA, 89, 12112-12116. MEDLINE Abstract

34. Yajima,H., Takao,M., Yasuhira,S., Zhao,J.H., Ishii,C., Inoue,H. and Yasui,A. (1995) EMBO J., 14, 2393-2399. MEDLINE Abstract

35. Doetsch,P.W. (1995) Trends Biochem. Sci., 20, 384-386. MEDLINE Abstract

36. McCready,S. and Cox,B. (1993) Mutat. Res., 293, 233-240. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +1 626 301 8879; Fax: +1 626 301 8972; Email: bshen@smtplink.coh.org

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