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• W1992475933 abstract "Ntg2p is a DNAN-glycosylase/apurinic or apyrimidinic lyase involved in base excision repair of oxidatively damaged DNA inSaccharomyces cerevisiae. Using a yeast two-hybrid screen and a GST in vitro transcription and translation assay, the mismatch repair (MMR) protein Mlh1p was demonstrated to interact physically with Ntg2p. The Mlh1p binding site maps to amino acids residues 15–40 of Ntg2p. The Ntg2p binding site is localized in the C-terminal end (483–769) of Mlh1p. Overproduction of Ntg2p results in a mutator phenotype with enhanced frameshift reversion frequency, suggesting partial inhibition of the MMR pathway. In contrast, inactivation of NTG2 does not enhance mutagenesis, indicating that Ntg2p is not required for MMR. Site-directed mutagenesis of the Mlh1p binding domain of Ntg2p revealed three amino acids (Ser24, Tyr26, Phe27) that are absolutely required for Ntg2p-Mlh1p interaction. These residues are part of a motif found in Ntg2p (Arg23-Ser24-Lys25-Tyr26-Phe27), Exo1p (Arg444-Ser445-Lys446-Phe447-Phe448), and Sgs1p (Lys1383-Ser1384-Lys1385-Phe1386-Phe1387). In these three proteins, the motif is part of the domain that interacts with the C-terminal end of Mlh1p. Furthermore, S445A, F447A, and F448A mutants of Exo1p do not bind Mlh1p, but the wild type Exo1p does. Therefore, we propose that the R/K-S-R/K-Y/F-Y/F sequence could define a Mhl1 binding motif. The results also suggest that base excision repair and MMR can cooperate to prevent deleterious effects of oxidative DNA damage. Ntg2p is a DNAN-glycosylase/apurinic or apyrimidinic lyase involved in base excision repair of oxidatively damaged DNA inSaccharomyces cerevisiae. Using a yeast two-hybrid screen and a GST in vitro transcription and translation assay, the mismatch repair (MMR) protein Mlh1p was demonstrated to interact physically with Ntg2p. The Mlh1p binding site maps to amino acids residues 15–40 of Ntg2p. The Ntg2p binding site is localized in the C-terminal end (483–769) of Mlh1p. Overproduction of Ntg2p results in a mutator phenotype with enhanced frameshift reversion frequency, suggesting partial inhibition of the MMR pathway. In contrast, inactivation of NTG2 does not enhance mutagenesis, indicating that Ntg2p is not required for MMR. Site-directed mutagenesis of the Mlh1p binding domain of Ntg2p revealed three amino acids (Ser24, Tyr26, Phe27) that are absolutely required for Ntg2p-Mlh1p interaction. These residues are part of a motif found in Ntg2p (Arg23-Ser24-Lys25-Tyr26-Phe27), Exo1p (Arg444-Ser445-Lys446-Phe447-Phe448), and Sgs1p (Lys1383-Ser1384-Lys1385-Phe1386-Phe1387). In these three proteins, the motif is part of the domain that interacts with the C-terminal end of Mlh1p. Furthermore, S445A, F447A, and F448A mutants of Exo1p do not bind Mlh1p, but the wild type Exo1p does. Therefore, we propose that the R/K-S-R/K-Y/F-Y/F sequence could define a Mhl1 binding motif. The results also suggest that base excision repair and MMR can cooperate to prevent deleterious effects of oxidative DNA damage. apurinic/apyrimidinic activation domain base excision repair canavanine resistance DNA binding domain 5,6-dihydrothymidine glutathioneS-transferase in vitrotranscription-translation 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine mismatch repair nucleotide excision repair 5-bromo- 4-chloro-3-indolyl-β-d-galactopyranoside Oxidative DNA damage has been involved in pathological processes such as cancer, neurodegenerative diseases, and aging (1Feig D.I. Reid T.M. Loeb L.A. Cancer Res. 1994; 54: 1890S-1894SPubMed Google Scholar, 2Ames B.N. Shigenaga M.K. Halliwell B. A. O. DNA and Free Radicals. Ellis Horwood, New York1993: 1-18Google Scholar, 3Wiseman H. Halliwell B. Biochem. J. 1996; 313: 17-29Crossref PubMed Scopus (1964) Google Scholar). It results from the attack of base and sugar moieties by reactive oxygen species generated by cellular metabolism or exogenous agents. Reactive oxygen species can induce several types of lesions in DNA, such as oxidized bases, apurinic/apyrimidinic (AP)1 sites and strand breakage (4Dizdaroglu M. Free Radic. Biol. Med. 1991; 10: 225-242Crossref PubMed Scopus (471) Google Scholar, 5Cadet J. Berger M. Douki T. Ravanat J.L. Rev. Physiol. Biochem. Pharmacol. 1997; 131: 1-87PubMed Google Scholar). To prevent the deleterious action of oxidative DNA damage, organisms have developed robust DNA repair mechanisms. Reactive oxygen species-induced lesions in DNA bases are primarily repaired by the base excision repair (BER) pathway. BER is a ubiquitous DNA repair process that is initiated by a DNA N-glycosylase removing the altered or unusual bases. Two classes of DNAN-glycosylases are present in the cell: the monofunctional enzymes catalyzing the removal of the lesion, leaving an AP site in DNA; and the DNA N-glycosylases/AP lyases catalyzing both cleavage of the N-glycosylic bond and of the phosphodiester bond 3′ to the newly formed AP site (6Sun B. Latham K.A. Dodson M.L. Lloyd R.S. J. Biol. Chem. 1995; 270: 19501-19508Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 7Cunningham R.P. Mutat. Res. 1997; 383: 189-196Crossref PubMed Scopus (96) Google Scholar). The next step in the course of BER is catalyzed by an AP endonuclease generating a single strand break with a 3′-OH end that can be used as a substrate by a DNA polymerase. Finally, BER is completed by successive action of a DNA polymerase and a DNA ligase (8Friedberg E. Walker G. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology Press, Washington, D. C.1995: 135-190Google Scholar, 9Seeberg E. Eide L. Bjoras M. Trends Biochem. Sci. 1995; 20: 391-397Abstract Full Text PDF PubMed Scopus (470) Google Scholar). In Saccharomyces cerevisiae, three DNAN-glycosylases/AP lyases are involved in the repair of oxidatively damaged DNA bases: Ntg1p, Ntg2p, and Ogg1p (10Girard P.M. Boiteux S. Biochimie (Paris). 1997; 79: 559-566Crossref PubMed Scopus (95) Google Scholar). Ntg1p and Ntg2p are closely related to each other and to Escherichia coli Nth (11Eide L. Bjoras M. Pirovano M. Alseth I. Berdal K.G. Seeberg E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10735-10740Crossref PubMed Scopus (146) Google Scholar, 12Augeri L. Lee Y.M. Barton A.B. Doetsch P.W. Biochemistry. 1997; 36: 721-729Crossref PubMed Scopus (79) Google Scholar, 13Senturker S. Auffret van der Kemp P. You H.J. Doetsch P.W. Dizdaroglu M. Boiteux S. Nucleic Acids Res. 1998; 26: 5270-5276Crossref PubMed Scopus (96) Google Scholar, 14Alseth I. Eide L. Pirovano M. Rognes T. Seeberg E. Bjoras M. Mol. Cell. Biol. 1999; 19: 3779-3787Crossref PubMed Google Scholar, 15Swanson R.L. Morey N.J. Doetsch P.W. Jinks-Robertson S. Mol. Cell. Biol. 1999; 19: 2929-2935Crossref PubMed Scopus (194) Google Scholar). However, Ntg2p, but not Ntg1p, possesses the consensus sequence for an iron-sulfur center found in most of the Nth homologs (11Eide L. Bjoras M. Pirovano M. Alseth I. Berdal K.G. Seeberg E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10735-10740Crossref PubMed Scopus (146) Google Scholar, 12Augeri L. Lee Y.M. Barton A.B. Doetsch P.W. Biochemistry. 1997; 36: 721-729Crossref PubMed Scopus (79) Google Scholar, 13Senturker S. Auffret van der Kemp P. You H.J. Doetsch P.W. Dizdaroglu M. Boiteux S. Nucleic Acids Res. 1998; 26: 5270-5276Crossref PubMed Scopus (96) Google Scholar, 14Alseth I. Eide L. Pirovano M. Rognes T. Seeberg E. Bjoras M. Mol. Cell. Biol. 1999; 19: 3779-3787Crossref PubMed Google Scholar, 15Swanson R.L. Morey N.J. Doetsch P.W. Jinks-Robertson S. Mol. Cell. Biol. 1999; 19: 2929-2935Crossref PubMed Scopus (194) Google Scholar). Cellular localization analysis indicates that Ntg2p is exclusively nuclear, whereas Ntg1p is both nuclear and mitochondrial (14Alseth I. Eide L. Pirovano M. Rognes T. Seeberg E. Bjoras M. Mol. Cell. Biol. 1999; 19: 3779-3787Crossref PubMed Google Scholar, 16You H.J. Swanson R.L. Harrington C. Corbett A.H. Jinks-Robertson S. Senturker S. Wallace S.S. Boiteux S. Dizdaroglu M. Doetsch P.W. Biochemistry. 1999; 38: 11298-11306Crossref PubMed Scopus (109) Google Scholar). Furthermore, Ntg1p is DNA damage-inducible, whereas Ntg2p is constitutively expressed (11Eide L. Bjoras M. Pirovano M. Alseth I. Berdal K.G. Seeberg E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10735-10740Crossref PubMed Scopus (146) Google Scholar, 12Augeri L. Lee Y.M. Barton A.B. Doetsch P.W. Biochemistry. 1997; 36: 721-729Crossref PubMed Scopus (79) Google Scholar, 14Alseth I. Eide L. Pirovano M. Rognes T. Seeberg E. Bjoras M. Mol. Cell. Biol. 1999; 19: 3779-3787Crossref PubMed Google Scholar). Ogg1p does not show significant sequence homology with Ntg1p and Ntg2p except for the helix-hairpin-helix-GPD/K active site domain (17Nash H.M. Bruner S.D. Scharer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, 18Guibourt N. Castaing B. Auffret van der Kemp P. Boiteux S. Biochemistry. 2000; 39: 1716-1724Crossref PubMed Scopus (22) Google Scholar, 19Auffret van der Kemp P. Thomas D. Barbey R. de Oliveira R. Boiteux S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5197-5202Crossref PubMed Scopus (348) Google Scholar). Ntg1p and Ntg2p display a broad substrate specificity, releasing oxidized pyrimidines (11Eide L. Bjoras M. Pirovano M. Alseth I. Berdal K.G. Seeberg E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10735-10740Crossref PubMed Scopus (146) Google Scholar, 12Augeri L. Lee Y.M. Barton A.B. Doetsch P.W. Biochemistry. 1997; 36: 721-729Crossref PubMed Scopus (79) Google Scholar, 13Senturker S. Auffret van der Kemp P. You H.J. Doetsch P.W. Dizdaroglu M. Boiteux S. Nucleic Acids Res. 1998; 26: 5270-5276Crossref PubMed Scopus (96) Google Scholar, 14Alseth I. Eide L. Pirovano M. Rognes T. Seeberg E. Bjoras M. Mol. Cell. Biol. 1999; 19: 3779-3787Crossref PubMed Google Scholar, 15Swanson R.L. Morey N.J. Doetsch P.W. Jinks-Robertson S. Mol. Cell. Biol. 1999; 19: 2929-2935Crossref PubMed Scopus (194) Google Scholar, 16You H.J. Swanson R.L. Harrington C. Corbett A.H. Jinks-Robertson S. Senturker S. Wallace S.S. Boiteux S. Dizdaroglu M. Doetsch P.W. Biochemistry. 1999; 38: 11298-11306Crossref PubMed Scopus (109) Google Scholar) but also purine-derived lesions (13Senturker S. Auffret van der Kemp P. You H.J. Doetsch P.W. Dizdaroglu M. Boiteux S. Nucleic Acids Res. 1998; 26: 5270-5276Crossref PubMed Scopus (96) Google Scholar). Although similar, Ntg1p and Ntg2p substrate specificities are not identical (13Senturker S. Auffret van der Kemp P. You H.J. Doetsch P.W. Dizdaroglu M. Boiteux S. Nucleic Acids Res. 1998; 26: 5270-5276Crossref PubMed Scopus (96) Google Scholar). Ogg1p exhibits a narrower substrate specificity, catalyzing the removal of two oxidized purines (20Karahalil B. Girard P.M. Boiteux S. Dizdaroglu M. Nucleic Acids Res. 1998; 26: 1228-1233Crossref PubMed Scopus (109) Google Scholar). Finally, Ntg1p, Ntg2p, and Ogg1p incise DNA at AP sites via a β-elimination reaction (14Alseth I. Eide L. Pirovano M. Rognes T. Seeberg E. Bjoras M. Mol. Cell. Biol. 1999; 19: 3779-3787Crossref PubMed Google Scholar, 20Karahalil B. Girard P.M. Boiteux S. Dizdaroglu M. Nucleic Acids Res. 1998; 26: 1228-1233Crossref PubMed Scopus (109) Google Scholar). The biological functions of Ntg1p, Ntg2p, and Ogg1p DNA N-glycosylases inS. cerevisiae have been investigated by analyzing the phenotypes of mutant strains. Recently, it has been reported that anntg1 ntg2 double mutant strain is not unusually sensitive to H2O2, nor does it exhibit a mutator phenotype (15Swanson R.L. Morey N.J. Doetsch P.W. Jinks-Robertson S. Mol. Cell. Biol. 1999; 19: 2929-2935Crossref PubMed Scopus (194) Google Scholar, 21Gellon L. Barbey R. Auffret van der Kemp P. Thomas D. Boiteux S. Mol. Gen. Genet. 2001; 265: 1087-1096Crossref PubMed Scopus (61) Google Scholar). However, a ntg1 ntg2 rad14triple mutant exhibits a mutator phenotype and enhanced sensitivity to the lethal effect of chemical oxidants compared with that of a wild type strain (21Gellon L. Barbey R. Auffret van der Kemp P. Thomas D. Boiteux S. Mol. Gen. Genet. 2001; 265: 1087-1096Crossref PubMed Scopus (61) Google Scholar). These results show that oxidatively damaged pyrimidines are not only removed by BER enzymes such as Ntg1p and Ntg2p but also by the nucleotide excision repair (NER) pathway. It was demonstrated recently that the Apn1p can also incise DNA on the 5′-side of various oxidatively damaged bases (22Ischenko A.A. Saparbaev M.K. Nature. 2002; 415: 183-187Crossref PubMed Scopus (218) Google Scholar). On the other hand, inactivation of the Ogg1p results in a spontaneous GC to TA mutator phenotype, which is probably caused by the accumulation of 8-oxoG in DNA (23Thomas D. Scot A.D. Barbey R. Padula M. Boiteux S. Mol. Gen. Genet. 1997; 254: 171-178Crossref PubMed Scopus (203) Google Scholar, 24Bruner S.D. Nash H.M. Lane W.S. Verdine G.L. Curr. Biol. 1998; 8: 393-403Abstract Full Text Full Text PDF PubMed Google Scholar). In S. cerevisiae, recent studies strongly suggest that the mutagenic action of endogenous oxidative DNA damage is also counteracted by the mismatch repair pathway (MMR) (25Earley M.C. Crouse G.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15487-15491Crossref PubMed Scopus (81) Google Scholar, 26Ni T.T. Marsischky G.T. Kolodner R.D. Mol. Cell. 1999; 4: 439-444Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 27Terato H. Masaoka A. Kobayashi M. Fukushima S. Ohyama Y. Yoshida M. Ide H. J. Biol. Chem. 1999; 274: 25144-25150Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 28Jackson A.L. Chen R. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12468-12473Crossref PubMed Scopus (194) Google Scholar). A genetic analysis showed that reversion rates for three classes of base substitutions are greatly enhanced in yeast strains deficient in Msh2p or Msh6p; however, when cells are grown anaerobically, the reversion rates for these base substitutions, in those strains, are strongly reduced (25Earley M.C. Crouse G.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15487-15491Crossref PubMed Scopus (81) Google Scholar). Moreover, ogg1 msh2 and ogg1 msh6double mutants exhibit a synergistic increase in GC to TA spontaneous transversions compared with the ogg1, msh2, and msh6 single mutants (26Ni T.T. Marsischky G.T. Kolodner R.D. Mol. Cell. 1999; 4: 439-444Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). These last results suggest that MMR eliminates adenine residues incorporated opposite 8-oxoG (26Ni T.T. Marsischky G.T. Kolodner R.D. Mol. Cell. 1999; 4: 439-444Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). MMR could also remove 8-oxoG incorporated by a DNA polymerase using 8-oxo-dGTP as a precursor in the course of DNA replication. Therefore, MMR could be the functional homolog in S. cerevisiae of the bacterial MutY and MutT (26Ni T.T. Marsischky G.T. Kolodner R.D. Mol. Cell. 1999; 4: 439-444Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 29Mazurek A. Berardini M. Fishel R. J. Biol. Chem. 2002; 277: 8260-8266Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Taken together, the results show that in S. cerevisiae, BER, NER, and MMR pathways are involved in the repair of oxidative DNA damage and therefore cooperate to prevent its deleterious action. The cooperation of these different pathways could be mediated by protein-protein interactions. To explore this avenue, we have performed a yeast two-hybrid screen using the full-length Ntg2p as bait and a library of genomic DNA fragments of S. cerevisiae as a source of potential protein partners. In this report, we demonstrate that Ntg2p specifically interacts with the MMR protein Mlh1p. In addition, overexpression of Ntg2p leads to a MMR mutator phenotype, whereas deletion of NTG2 has no effect on MMR. Finally, we could determine the presence of an Mlh1p interaction motif in partners of Mlh1p such as Ntg2p, Sgs1p, and Exo1p. Microbiological techniques and media used in this study have been described by Miller (30Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar) and Sherman (31Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2543) Google Scholar) for E. coli and S. cerevisiae, respectively. Bacterial strains DH5α and JM105 were used for plasmid construction and preparation (32Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1982Google Scholar). Yeast two-hybrid tests were performed in strain Y190 (MATa gal4 gal80 his3 trp1–901 ade2–101 ura3–52 leu2–3, 112 URA3::GAL1::lacZ LYS2::GAL4(UAS)::HIS3 cyhR) (33Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5235) Google Scholar). Other S. cerevisiaestrains used in this study are listed in Table I. Deletion mutants ofMLH1, PMS1, MSH2, NTG2, and EXO1 were constructed by the PCR-mediated one-step replacement technique using the kanMX module (34Baudin A. Ozier-Kalogeropoulos O. Denouel A. Lacroute F. Cullin C. Nucleic Acids Res. 1993; 21: 3329-3330Crossref PubMed Scopus (1119) Google Scholar, 35Longtine M.S. McKenzie III, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4169) Google Scholar). Yeast transformations were performed using the polyethylene glycol/lithium method (36Gietz D., St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2893) Google Scholar). Gene replacement was confirmed by PCR analysis on genomic DNA.Table IS. cerevisiae strains used in this studyStrainGenotypeSource/referenceRKY3109MATa ura3–52, leu2Δ1, trp1Δ63, his3Δ200, lys2ΔBgl, hom3–10, ade2Δ1, ade8P. BertrandBG301RKY3109 with mlh1Δ∷kanMX6This workBG307RKY3109 with exo1Δ∷kanMX6This workBG309RKY3109 with ntg2Δ∷kanMX6This workBG312RKY3109 with mlh1Δ∷kanMX6, ntg2Δ∷TRP1This workBG308Y190 with pms1Δ∷kanMX6This workBG313Y190 with msh2Δ∷kanMX6This work Open table in a new tab Full-length and truncatedNTG1, NTG2, MLH1, and EXO1open reading frames were prepared by PCR amplification from yeast genomic DNA using the Pfu DNA polymerase (Stratagene) and specific primers (Genosys) harboring the BamHI and SmaI restriction sites allowing in-frame insertion of the open reading frames into the vectors. Yeast two-hybrid plasmid vectors are pGBT9 (37Bartel P. Chien C.T. Sternglanz R. Fields S. BioTechniques. 1993; 14: 920-924PubMed Google Scholar) and pAS2ΔΔ (38Fromont-Racine M. Rain J.C. Legrain P. Nat. Genet. 1997; 16: 277-282Crossref PubMed Scopus (711) Google Scholar) yielding bait constructs and pACT2 for prey constructs. Bait constructs express Gal4pDBD(DNA Binding Domain) C-terminal fusion proteins. Prey constructs express Gal4pAD(Activation Domain) C-terminal fusion proteins. Plasmids pGEX4T-1 (Amersham Biosciences) and pBluescript II KS (Stratagene) were used for GST-in vitrotranscription-translation (IVTT) assays. Plasmid PTRC99a (AmershamBiosciences) was used to express and purify full-length Ntg2p and truncated Ntg2p(31–380). Finally, pYX212 (R&D) was used to express native Ntg2p. All constructs were confirmed by sequencing. Sequences of plasmids, construction schemes, and oligonucleotide primers are available upon request. Point mutations were performed by PCR-mediated mutagenesis (QuikChange site-directed mutagenesis Kit, Stratagene). Alanine substitutions at the N-terminal end of Ntg2p are generated using pAS2ΔΔ-NTG2 as target and specific primers. The plasmid expressing Gal4pDBD-Ntg2p(1–20) was constructed by changing Gln21/Val22/Arg23 of Ntg2p into three stop codons. The plasmid expressing Gal4pAD-Mlh1p(1–731) was constructed by changing Glu732/His733/Val734 into stop codons. All constructs were confirmed by sequencing. Sequences of plasmids, construction schemes, and oligonucleotides primers are available upon request. Yeast two-hybrid screening was performed with the FRYL genomic library (38Fromont-Racine M. Rain J.C. Legrain P. Nat. Genet. 1997; 16: 277-282Crossref PubMed Scopus (711) Google Scholar) containing randomly sheared genomic DNA fragments of 700 bp mean size in a modified pACT2 vector. The library DNA was purified by ultracentrifugation in a CsCl gradient to transform strain Y190 containing the Gal4pDBD-Ntg2p bait fusion. Transformed cells were plated directly onto 100 μg/ml SD adenine minimal medium plates containing 25 or 50 mm 3-aminotriazole to select His+ colonies. Colonies growing after 4–7 days were streaked on SD + adenine + histidine and assayed for β-galactosidase production in an overlay plate assay. Blue colonies were restreaked on the same medium and tested again for β-galactosidase production. Among more than 1.1 × 107 transformants tested for histidine prototrophy and positive β-galactosidase assays, positive clones were selected. Prey plasmid DNA was recovered and transformed in E. coli strain 1066 (trpC-9830 leuB-6 pyrF-74::Tn5Δ(lac I POZYA)-74 galU galK hsdR rpsL) selecting the yeast LEU2 marker. Alternatively, we prepared total yeast DNA by using a QIAamp kit (Qiagen, Chatsworth, CA), amplified the prey DNA by PCR, and purified the product on a QIAquick column. After sequencing, the identity of the insert was determined by using the S. cerevisiae Genome Data base Blast service (genome-www2.stanford.edu/cgi-bin/SGD/nph-blast2sgd). β-Galactosidase liquid assays were performed by resuspending cells in 1 ml of Z buffer with 0.8 mg of o-nitrophenyl β-d-galactopyranoside substrate plus chloroform and SDS. Reaction were stopped with Na2CO3 when an appropriate level of color had developed, and β-galactosidase activity was measured (30Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar). The average and S.D. of at least three independent experiments are presented. GST and GST-Ntg2p were expressed in E. coli JM105 harboring a pGEX4T-1 or pGEX-NTG2, respectively. Cells were grown at 37 °C in LB broth medium (1 liter) containing 100 μg/ml ampicillin, until theA600 = 0.7 and induced for 16 h at 20 °C in the presence of 0.1 mmisopropyl-1-thio-β-d-galactopyranoside. Cells were collected and stored at –80 °C. Cell pellets were resuspended into 10 ml/g lysis buffer (20 mm Tris-HCl, pH 8.2, 1 mm Na2EDTA, 500 mm NaCl, 0.8 μg/ml antipain, 0.8 μg/ml leupeptine, 0.8 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride). Cell suspensions were sonicated and centrifuged at 4 °C. The supernatant fractions were dialyzed against phosphate-buffered saline buffer and applied to glutathione-Sepharose 4B (Amersham Biosciences) equilibrated with phosphate-buffered saline buffer for 1 h at room temperature under gentle continuous agitation. [35S]Methionine-labeled Mlh1p was prepared by IVTT of pBluescript II KS-MLH1 using a TNT-coupled lysate system (Promega) and [35S]methionine (Amersham Biosciences). 35 μl of [35S]methionine-labeled Mlh1p was added to equivalent amounts of GST or GST-Ntg2p bound to the glutathione-Sepharose 4B beads in 200 μl of buffer (50 mm Tris-HCl, pH 7.5, 0.1% Nonidet P-40, 150 mm NaCl, 1 mmNa2EDTA, 0.3 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride). The mixture was incubated for 15 min at 37 °C on a rotator. Beads were washed three times with wash buffer (50 mm Tris-HCl, pH 7.5, 0.1% Nonidet P-40, 200 mm NaCl, 1 mmNa2EDTA, 0.3 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride). Beads were pelleted and resuspended in denaturing loading buffer, boiled, and analyzed by 10% SDS-PAGE, and imaged using a Storm PhosphorImager (Molecular Dynamics). Wild type cells containing the Gal4pDBD-tagged baits were grown in selective medium to exponential phase before extraction. Yeast extracts were prepared as described by Vialard et al. (39Vialard J.E. Gilbert C.S. Green C.M. Lowndes N.F. EMBO J. 1998; 17: 5679-5688Crossref PubMed Scopus (228) Google Scholar) by glass bead beating in 20% trichloroacetic acid, washing the glass beads in 5% trichloroacetic acid, and combining the wash with the lysate. The protein suspension was then pelleted, resuspended in Laemmli loading buffer (pH 8.8), boiled for 5 min, pelleted, and the supernatant was retained as a whole cell extract. For Western blotting, proteins were separated on 12.5% SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences) by electroblotting (Bio-Rad). Monoclonal antibody raised against Gal4pDBD(CLONTECH) was incubated with the nitrocellulose membranes as recommended in the manufacturer's protocol. Secondary horseradish peroxidase-conjugated anti-mouse antibody (AmershamBiosciences) was incubated for 1 h at a 1:10,000 dilution, and the blot was revealed by chemiluminescence (Amersham Biosciences). The 34-mer oligodeoxyribonucleotide used in this study was 5′-GGCTTCATCGTTATC(DHT)CTGACCTGGTGGATACCG-3′. DHT represents a site-specifically incorporated 5,6-dihydrothymidine residue. The DHT-containing strand was 32P-labeled at the 5′-end and annealed with a complementary strand containing an adenine opposite DHT as described (40D'Ham C. Romieu A. Jaquinod M. Gasparutto D. Cadet J. Biochemistry. 1999; 38: 3335-3344Crossref PubMed Scopus (97) Google Scholar). Crude extract was obtained from yeast Y190-harboring plasmids expressing wild type or mutant Gal4pDBD-Ntg2p as described by Thomas et al. (23Thomas D. Scot A.D. Barbey R. Padula M. Boiteux S. Mol. Gen. Genet. 1997; 254: 171-178Crossref PubMed Scopus (203) Google Scholar). In a standard reaction (20-μl final volume), 50 fmol of32P-labeled DHT·A duplex was incubated in reaction buffer (25 mm Tris-HCl, pH 7.6, 5 mmNa2EDTA) with cell-free protein extracts (3.5 μg of protein). Reactions were carried out at 37 °C for 30 min and stopped by adding 5 μl of formamide dye and subjected to 7 m urea and 20% PAGE as described by Girard et al. (41Girard P.M. D'Ham C. Cadet J. Boiteux S. Carcinogenesis. 1998; 19: 1299-1305Crossref PubMed Scopus (73) Google Scholar). Gels were scanned and band intensities quantified using a Molecular Dynamics PhosphorImager. 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimide (Me-FapyG)-containing DNA was also used as substrate. For the Me-FapyG DNA N-glycosylase activity assay, [3H]Me-FapyG-poly(dG-dC) was prepared as described (42Boiteux S. Belleney J. Roques B.P. Laval J. Nucleic Acids Res. 1984; 12: 5429-5439Crossref PubMed Scopus (99) Google Scholar). Full-length Ntg2p was purified to homogeneity as described previously (13Senturker S. Auffret van der Kemp P. You H.J. Doetsch P.W. Dizdaroglu M. Boiteux S. Nucleic Acids Res. 1998; 26: 5270-5276Crossref PubMed Scopus (96) Google Scholar). The same strategy was used to purify truncated Ntg2p(31–380). The assay mixture (100 μl) was composed of 25 mmTris-HCl, pH 7.6, 100 mm KCl, [3H]Me-FapyG-poly(dG-dC), and purified protein. The reaction was carried out at 37 °C for 15 min, and the ethanol-soluble radioactive material was quantitated. Spontaneous reversion of the hom3–10 or lys2-Bgl locus and forward mutation to canavanine resistance (CanR), were measured. At least 10 independent yeast cultures were started from about 103 viable cells and grown to stationary phase in liquid YPAD medium or YNBD medium supplemented with required base or amino acids. Cells were plated after appropriate dilution or concentration onto selective medium plates lacking threonine or lysine for Hom+ and Lys+ revertants or containing 60 μg/ml l-canavanine for CanR mutants. Plates were incubated for 3–5 days at 30 °C before counting. Spontaneous mutant frequencies were calculated by dividing the revertant or mutant count by the viable cell count. Values were determined by the method of the median (43Lea D.E. Coulson C.A. J. Genet. 1949; 49: 264-285Crossref PubMed Scopus (1082) Google Scholar). S.D. values were calculated, and the 95% confidence interval was applied by using PRISM 2.0 software (GraphPad, San Diego). Using full-length Ntg2p (Fig.1A) as bait in a two-hybrid screen of 1.1 × 107 transformants, 15 positive colonies were selected and analyzed by sequencing. The three strongest interactions, based on the early appearance of colonies on (His− + 3-aminotriazole) plates and on the intensity of color in the X-gal overlay plate assay, were found to match two fragments of the C-terminal end (402–769) and (483–769), from the MMR protein, Mlh1p (Fig. 1, B and C). Interestingly, the C-terminal end of Mlh1p also interacts with Pms1p, Mlh2p, Mlh3p, Exo1p, or Sgs1p in two-hybrid assays (44Pang Q. Prolla T.A. Liskay R.M. Mol. Cell. Biol. 1997; 17: 4465-4473Crossref PubMed Scopus (115) Google Scholar, 45Wang T.F. Kleckner N. Hunter N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13914-13919Crossref PubMed Scopus (239) Google Scholar, 46Tran P.T. Simon J.A. Liskay R.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9760-9765Crossref PubMed Scopus (107) Google Scholar). Because the Mlh1p preys were truncated, it was important to test the ability of full-length Mlh1p to interact with Ntg2p. Fig. 1, B and C, shows that the full-length Mlh1p interacts with Ntg2p in a two-hybrid assay as well as the truncated Mlh1p(483–769) does. Because Ntg2p and Ntg1p present a high degree of similarity, we also performed a two-hybrid assay with full-length Ntg1p as bait and full-length Mlh1p as prey. Fig. 1B shows that Ntg1p does not interact with Mlh1p by two-hybrid assay. We also constructed a Mlh1p(1–731) mutant with its 38 last amino acids deleted and tested it for interaction with the full-length Ntg2p by the two-hybrid system. Fig. 1Cshows that the Mlh1p(1–731) does not interact with Ntg2p. Control experiments show that neither Ntg2p bait nor full-length or truncated Mlh1p preys alone stimulate reporter gene expression" @default.
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• W1992475933 title "Ntg2p, a Saccharomyces cerevisiae DNAN-Glycosylase/Apurinic or Apyrimidinic Lyase Involved in Base Excision Repair of Oxidative DNA Damage, Interacts with the DNA Mismatch Repair Protein Mlh1p" @default.
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• W1992475933 doi "https://doi.org/10.1074/jbc.m202963200" @default.
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