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hUNG2 Is the Major Repair Enzyme for Removal of Uracil from U:A Matches, U:G Mismatches, and U in Single-stranded DNA, with hSMUG1 as a Broad Specificity Backup

2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês

10.1074/jbc.m207107200

ISSN

1083-351X

Autores

Bodil Kavli, Ottar Sundheim, Mansour Akbari, Marit Otterlei, Hilde Nilsen, Frank Skorpen, Per Arne, Lars Hagen, Hans E. Krokan, Geir Slupphaug,

Tópico(s)

Biochemical and Molecular Research

Resumo

hUNG2 and hSMUG1 are the only known glycosylases that may remove uracil from both double- and single-stranded DNA in nuclear chromatin, but their relative contribution to base excision repair remains elusive. The present study demonstrates that both enzymes are strongly stimulated by physiological concentrations of Mg2+, at which the activity of hUNG2 is 2–3 orders of magnitude higher than of hSMUG1. Moreover, Mg2+ increases the preference of hUNG2 toward uracil in ssDNA nearly 40-fold. APE1 has a strong stimulatory effect on hSMUG1 against dsU, apparently because of enhanced dissociation of hSMUG1 from AP sites in dsDNA. hSMUG1 also has a broader substrate specificity than hUNG2, including 5-hydroxymethyluracil and 3,N 4-ethenocytosine. hUNG2 is excluded from, whereas hSMUG1 accumulates in, nucleoli in living cells. In contrast, only hUNG2 accumulates in replication foci in the S-phase. hUNG2 in nuclear extracts initiates base excision repair of plasmids containing either U:A and U:G in vitro. Moreover, an additional but delayed repair of the U:G plasmid is observed that is not inhibited by neutralizing antibodies against hUNG2 or hSMUG1. We propose a model in which hUNG2 is responsible for both prereplicative removal of deaminated cytosine and postreplicative removal of misincorporated uracil at the replication fork. We also provide evidence that hUNG2 is the major enzyme for removal of deaminated cytosine outside of replication foci, with hSMUG1 acting as a broad specificity backup. hUNG2 and hSMUG1 are the only known glycosylases that may remove uracil from both double- and single-stranded DNA in nuclear chromatin, but their relative contribution to base excision repair remains elusive. The present study demonstrates that both enzymes are strongly stimulated by physiological concentrations of Mg2+, at which the activity of hUNG2 is 2–3 orders of magnitude higher than of hSMUG1. Moreover, Mg2+ increases the preference of hUNG2 toward uracil in ssDNA nearly 40-fold. APE1 has a strong stimulatory effect on hSMUG1 against dsU, apparently because of enhanced dissociation of hSMUG1 from AP sites in dsDNA. hSMUG1 also has a broader substrate specificity than hUNG2, including 5-hydroxymethyluracil and 3,N 4-ethenocytosine. hUNG2 is excluded from, whereas hSMUG1 accumulates in, nucleoli in living cells. In contrast, only hUNG2 accumulates in replication foci in the S-phase. hUNG2 in nuclear extracts initiates base excision repair of plasmids containing either U:A and U:G in vitro. Moreover, an additional but delayed repair of the U:G plasmid is observed that is not inhibited by neutralizing antibodies against hUNG2 or hSMUG1. We propose a model in which hUNG2 is responsible for both prereplicative removal of deaminated cytosine and postreplicative removal of misincorporated uracil at the replication fork. We also provide evidence that hUNG2 is the major enzyme for removal of deaminated cytosine outside of replication foci, with hSMUG1 acting as a broad specificity backup. uracil-DNA glycosylase base excision repair AP endonuclease proliferating cell nuclear antigen polymerase enhanced yellow fluorescent protein enhanced cyan fluorescent protein fluorouracil 3,N 4-ethenocytosine single-stranded double-stranded nuclear localization signal hydroxymethyluracil dithiothreitol enhanced green fluorescent protein bovine serum albumin 5-methylcytosine Uracil in DNA can be introduced via two mechanisms, deamination of cytosine and misincorporation of dUMP during replication. Deamination of cytosine has been calculated from measured deamination rates to occur at a rate of 100–500 per human cell/day (1Frederico L.A. Kunkel T.A. Shaw B.R. Biochemistry. 1990; 29: 2532-2537Crossref PubMed Scopus (398) Google Scholar, 2Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4299) Google Scholar) to yield mutagenic U:G mispairs. Uracil may also appear as a consequence of misincorporation of dUMP instead of dTMP during replication, resulting in a U:A base pair. The latter is not miscoding, but may produce cytotoxic and mutagenic AP site intermediates during repair. In organisms containing 5-methylcytosine in their genomes, deamination of 5-methylcytosine furthermore leads to T:G mismatches. All living organisms express uracil-DNA glycosylases (UDGs)1 that prevent cytotoxic and mutagenic effects of the above lesions. UDGs remove uracil (and sometimes other damaged bases or thymine) from the deoxyribose and thus initiate a multistep base excision repair (BER) pathway, eventually restoring the correct DNA sequence. After removal of uracil by an UDG and cleavage of the resulting abasic site by AP endonuclease (APE1/APE2), the BER pathway splits into two branches (reviewed in Ref. 3Dogliotti E. Fortini P. Pascucci B. Parlanti E. Prog. Nucleic Acids Res. Mol. Biol. 2001; 68: 3-27Crossref PubMed Google Scholar). The presumed major track is the short-patch pathway. It uses the 5′- deoxyribophosphodiesterase activity of DNA polymerase β to cleave 3′ of the abasic site, thus releasing deoxyribose-5-phosphate. Then pol β inserts C or T, depending on the template base. Finally, DNA ligase III seals the nick, perhaps aided by the scaffold protein XRCC1. The alternative long-patch pathway largely uses replication proteins and may take place in replication foci (4Otterlei M. Warbrick E. Nagelhus T.A. Haug T. Slupphaug G. Akbari M. Aas P.A. Steinsbekk K. Bakke O. Krokan H.E. EMBO J. 1999; 18: 3834-3844Crossref PubMed Scopus (295) Google Scholar). This pathway requires pol ε and/or δ, as well as the trimeric sliding clamp and polymerase processivity factor proliferating cell nuclear antigen (PCNA) and the clamp loader replication factor C (RFC). Repair synthesis is stimulated by pol β, which may be important in the first step of polymerization. The structure-specific endonuclease FEN1 removes the 2–8-nucleotide displaced “flap” of DNA, and DNA ligase I seals the nick (3Dogliotti E. Fortini P. Pascucci B. Parlanti E. Prog. Nucleic Acids Res. Mol. Biol. 2001; 68: 3-27Crossref PubMed Google Scholar). Mammalian cells contain at least four UDGs, of which three (UNG, SMUG1, and TDG) belong to the same protein superfamily, possess the same fold, and have probably evolved from a common ancestor (5Aravind L. Koonin E.V. Genome Biol. 2000; 1: 7.1-7.8Crossref Google Scholar). Of these, UNG appears to be quantitatively dominating as determined from activity assays using human cell-free extracts and U:A substrates (6Slupphaug G. Eftedal I. Kavli B. Bharati S. Helle N.M. Haug T. Levine D.W. Krokan H.E. Biochemistry. 1995; 34: 128-138Crossref PubMed Scopus (246) Google Scholar). UNG belongs to the family of highly conserved UDGs typified byEscherichia coli Ung, and is present in a large number of eukaryotes, bacteria, and large eukaryotic DNA viruses (7Krokan H.E. Standal R. Slupphaug G. Biochem. J. 1997; 325: 1-16Crossref PubMed Scopus (725) Google Scholar). The human and mouse UNG genes encode both mitochondrial (UNG1) and nuclear (UNG2) forms of the enzyme by way of alternative promoter usage and mRNA splicing (8Nilsen H. Otterlei M. Haug T. Solum K. Nagelhus T.A. Skorpen F. Krokan H.E. Nucleic Acids Res. 1997; 25: 750-755Crossref PubMed Scopus (255) Google Scholar). The catalytic domain of hUNG has been extensively studied, and its structure and molecular mechanism of catalysis and specificity established (9Mol C.D. Arvai A.S. Slupphaug G. Kavli B. Alseth I. Krokan H.E. Tainer J.A. Cell. 1995; 80: 869-878Abstract Full Text PDF PubMed Scopus (340) Google Scholar, 10Slupphaug G. Mol C.D. Kavli B. Arvai A.S. Krokan H.E. Tainer J.A. Nature. 1996; 384: 87-92Crossref PubMed Scopus (483) Google Scholar, 11Parikh S.S. Walcher G. Jones G.D. Slupphaug G. Krokan H.E. Blackburn G.M. Tainer J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5083-5088Crossref PubMed Scopus (242) Google Scholar, 12Kavli B. Slupphaug G. Mol C.D. Arvai A.S. Petersen S.B. Tainer J.A. Krokan H.E. EMBO J. 1996; 15: 3442-3447Crossref PubMed Scopus (150) Google Scholar). The enzyme removes uracil in vitro in the order of preference ssU > U:G > U:A (6Slupphaug G. Eftedal I. Kavli B. Bharati S. Helle N.M. Haug T. Levine D.W. Krokan H.E. Biochemistry. 1995; 34: 128-138Crossref PubMed Scopus (246) Google Scholar). Certain closely related bases formed from cytosine after γ-irradiation or oxidative stress are also substrates, such as 5-hydroxyuracil, isodialuric acid, and alloxan (13Dizdaroglu M. Karakaya A. Jaruga P. Slupphaug G. Krokan H.E. Nucleic Acids Res. 1996; 24: 418-422Crossref PubMed Google Scholar). These are, however, excised at a very low rate compared with uracil. A second human UDG against both ss- and dsU, hSMUG1 (single-strand selective monofunctional uracil-DNA glycosylase), was identified more recently byin vitro expression cloning (14Haushalter K.A. Todd Stukenberg M.W. Kirschner M.W. Verdine G.L. Curr. Biol. 1999; 9: 174-185Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). SMUG1 is not found in bacteria and yeast, but is present in higher eukaryotes. Although they probably share the same fold and motifs necessary for substrate binding and catalysis as hUNG, little homology exists between the two enzymes at the amino acid level (5Aravind L. Koonin E.V. Genome Biol. 2000; 1: 7.1-7.8Crossref Google Scholar). hSMUG1 is furthermore located in the nuclei, and Xenopus SMUG1 has a substrate preference similar to the catalytic hUNG domain (ssU > U:G > U:A), although its specific activity is considerably lower (14Haushalter K.A. Todd Stukenberg M.W. Kirschner M.W. Verdine G.L. Curr. Biol. 1999; 9: 174-185Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Recently, Boorstein and co-workers (15Boorstein R.J. Cummings Jr., A. Marenstein D.R. Chan M.K., Ma, Y. Neubert T.A. Brown S.M. Teebor G.W. J. Biol. Chem. 2001; 276: 41991-41997Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) demonstrated that hSMUG1 is also active against 5-hydroxymethyluracil (HmU:A). HmU is formed in DNA by oxidative attack on the methyl group of thymine, thereby creating HmU:A. It is also the product of the deamination of 5-hydroxymethylcytosine, which may be formed via oxidation of 5-methylcytosine. The latter creates a HmU:G base pair, which would be mutagenic if left unrepaired. Although HmU:G substrates were not tested, the authors suggested the latter could be a biologically important substrate (15Boorstein R.J. Cummings Jr., A. Marenstein D.R. Chan M.K., Ma, Y. Neubert T.A. Brown S.M. Teebor G.W. J. Biol. Chem. 2001; 276: 41991-41997Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). This is also supported by the fact that transition mutation from 5mC:G to T:A is the most frequent substitution mutation in human cancer (16Cooper D.N. Youssoufian H. Hum. Genet. 1988; 78: 151-155Crossref PubMed Scopus (791) Google Scholar). The two last UDGs identified in human cells, TDG and MBD4 (MED1), are both mismatch-specific and have no activity against single-stranded substrates. TDG excises uracil and thymine from U:G and T:G mismatches, as well as 3,N 4-ethenocytosine (εC) and 5-fluorouracil (5-FU) from double-stranded DNA, and may restore G:C base pairs at sites of cytosine or 5-methylcytosine deamination, or alkylation, respectively (17Hardeland U. Bentele M. Lettieri T. Steinacher R. Jiricny J. Schar P. Prog. Nucleic Acids Res. Mol. Biol. 2001; 68: 235-253Crossref PubMed Google Scholar). MBD4, which does not belong to the same superfamily as the three other UDGs, acts on uracil, thymine, 5-FU, and εC mispaired with guanine (18Bellacosa A. J. Cell. Physiol. 2001; 187: 137-144Crossref PubMed Scopus (88) Google Scholar), as well as on 5-methylcytosine at hemimethylated DNA (19Zhu B. Zheng Y. Angliker H. Schwarz S. Thiry S. Siegmann M. Jost J.P. Nucleic Acids Res. 2000; 28: 4157-4165Crossref PubMed Scopus (152) Google Scholar). The preferred substrates, however, are G:T mismatches at methylated or unmethylated CpG islands. Thus, MBD4, as well as TDG, may have a function in the correction of T:G mismatches originating from deamination of 5-methylcytosine. Several lines of evidence indicate that nuclear UNG2 has a major role in postreplicative removal of misincorporated uracil in mammalian cells (4Otterlei M. Warbrick E. Nagelhus T.A. Haug T. Slupphaug G. Akbari M. Aas P.A. Steinsbekk K. Bakke O. Krokan H.E. EMBO J. 1999; 18: 3834-3844Crossref PubMed Scopus (295) Google Scholar). The contribution of UNG2 to repair of deaminated cytosines has, however, been debated. Whereas bacterial and yeastung − mutants display a mutator phenotype unable to repair deaminated cytosines (20Duncan B.K. Weiss B. J. Bacteriol. 1982; 151: 750-755Crossref PubMed Google Scholar, 21Impellizzeri K.J. Anderson B. Burgers P.M. J. Bacteriol. 1991; 173: 6807-6810Crossref PubMed Google Scholar), such a phenotype is not clearly observed in Ung −/− mice (22Nilsen H. Rosewell I. Robins P. Skjelbred C.F. Andersen S. Slupphaug G. Daly G. Krokan H.E. Lindahl T. Barnes D.E. Mol. Cell. 2000; 5: 1059-1065Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Based on this finding, and comparison of kinetic parameters of theXenopus SMUG1 (14Haushalter K.A. Todd Stukenberg M.W. Kirschner M.W. Verdine G.L. Curr. Biol. 1999; 9: 174-185Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) and the hUNG catalytic domain (6Slupphaug G. Eftedal I. Kavli B. Bharati S. Helle N.M. Haug T. Levine D.W. Krokan H.E. Biochemistry. 1995; 34: 128-138Crossref PubMed Scopus (246) Google Scholar), it was suggested that in higher eukaryotes, the contribution of UNG2 to the excision of deaminated cytosines was reduced, and that this function might instead be provided by SMUG1 (23Nilsen H. Haushalter K.A. Robins P. Barnes D.E. Verdine G.L. Lindahl T. EMBO J. 2001; 20: 4278-4286Crossref PubMed Scopus (161) Google Scholar). In human cells, however, the UNG catalytic domain alone is not observed in the nucleus (24Muller-Weeks S. Mastran B. Caradonna S. J. Biol. Chem. 1998; 273: 21909-21917Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Rather, the entire N-terminal regulatory domain remains attached to the core catalytic domain after nuclear translocation. Until now, little information has existed on the enzymatic properties of full-length hUNG2. A likely reason for this is the susceptibility of the enzyme to N-terminal proteolytic degradation during purification, mainly resulting in the core catalytic domain (25Wittwer C.U. Bauw G. Krokan H.E. Biochemistry. 1989; 28: 780-784Crossref PubMed Scopus (37) Google Scholar). To gain further insight in the functional properties of hUNG2 and hSMUG1 and their relative contribution to nuclear base excision repair, both proteins were purified after overexpression in E. coli. In depth biochemical characterization and analysis of subnuclear localization revealed previously unrecognized properties of both hUNG2 and hSMUG1 that may have important implications for their functions in nuclear BER in vivo. AnNdeI site was introduced at the hUNG2 translation start codon, and a 947-bp NdeI/HpaI fragment encompassing the entire hUNG2 coding region was ligated into the new vector pJB658cop251kan. This expression vector was constructed by introducing a high copy number variant of thetrfA gene (Arg-251 → Met) and changing the resistance from ampicillin to kanamycin in the toluic acid-inducible, broad host vector pJB658 (26Blatny J.M. Brautaset T. Winther-Larsen H.C. Karunakaran P. Valla S. Plasmid. 1997; 38: 35-51Crossref PubMed Scopus (136) Google Scholar). The resultant construct, p658KanUng2 was introduced intoE. coli BL21. Fermentation, preparation of crude extract, and the initial chromatographic steps were essentially as described for the hUNG catalytic domain (6Slupphaug G. Eftedal I. Kavli B. Bharati S. Helle N.M. Haug T. Levine D.W. Krokan H.E. Biochemistry. 1995; 34: 128-138Crossref PubMed Scopus (246) Google Scholar) except that the culture was induced by 1 mm toluic acid (final) and allowed to grow for additionally 3 h at 30 °C before harvesting. Furthermore, Complete® mini (EDTA-free) (Roche) protease inhibitor tablets were included in the initial steps of the purification (1 tablet/10 ml during homogenization and 1 tablet/l ml in subsequent buffers). During size exclusion chromatography on Superdex 75 26/60 (Amersham Biosciences), buffer was changed to 20 mmHEPES-NaOH (pH 8.0), 100 mm NaCl, 1 mm DTT. Pooled active fractions were loaded onto a MonoS HR 5/5 column, and after washing to base-line absorbance, bound proteins were eluted using a linear NaCl gradient from 100 mm to 1 m NaCl in the same buffer. To remove partially N-terminally deleted species, the purest fractions were subjected to MonoS rechromatography and fractions containing the highest portion of apparently full-length protein were pooled. The purified protein was finally concentrated by ultrafiltration and snap-frozen in liquid N2 prior to storage at −80 °C. IMAGE clone identification no. 726197 containing the hSMUG1 cDNA in pT7T3D was cut using NdeI and BfaI, and the 1154-bp fragment encoding hSMUG1 was cloned into the NdeI site of pET11a (Invitrogen). The resultant vector was transformed intoE. coli BL21-CodonPlus(DE3)-RIL (Stratagene) and bacterial cell mass for purification produced by fermentation. Expression was induced by 1 mmisopropyl-1-thio-β-d-galactopyranoside (final) at 37 °C and the culture allowed to grow for additional 4 h prior to cell harvest. Preparation of the hSMUG1 crude extract was essentially as described for the hUNG catalytic domain (6Slupphaug G. Eftedal I. Kavli B. Bharati S. Helle N.M. Haug T. Levine D.W. Krokan H.E. Biochemistry. 1995; 34: 128-138Crossref PubMed Scopus (246) Google Scholar), except that homogenization and protamine sulfate precipitation was performed in 20 mm HEPES-NaOH (pH 6.8), 50 mm NaCl, 0.1 mm EDTA, 1 mm DTT, and 1 tablet/10 ml Complete® mini (EDTA-free) protease inhibitor mixture (buffer A). The protamine sulfate fraction was loaded onto a DEAE-Sephacel column (Amersham Biosciences, 5 × 9 cm) coupled in series with a CM-Sepharose column (Amersham Biosciences, 5 × 8.5 cm). After washing to base-line absorbance, the DEAE-Sephacel column was bypassed and adsorbed proteins were eluted in a 0.05–1m NaCl gradient in buffer A. Active fractions were pooled and dialyzed against 20 mm HEPES-NaOH (pH 8), 10 mm NaCl, 1 mm DTT (buffer B) and applied to a UnoS-12 column (Bio-Rad). Adsorbed proteins were eluted in a 0.01–0.7m linear NaCl gradient. Active fractions were pooled and concentrated by ultrafiltration to 5 ml and loaded onto a Superdex 75 HiLoad 26/60 column (Amersham Biosciences) preequilibrated with buffer B containing 100 mm NaCl, and eluted with the same buffer. Fractions containing hSMUG1 were verified by SDS-PAGE and pooled. After a 4-fold dilution in buffer B, hSMUG1 was loaded onto a poly(U)-Sepharose column (Amersham Biosciences, 1.6 × 10 cm) preequilibrated with buffer B containing 2 mm EDTA. Adsorbed proteins were eluted in a 0.01–1 m linear NaCl gradient in the same buffer and the fractions containing hSMUG1 identified by SDS-PAGE and pooled. The poly(U)-Sepharose fraction was then dialyzed against buffer B, applied onto a MonoS HR 5/5 column, and eluted in a linear 0.01–1 m NaCl gradient in the same buffer. The purest hSMUG1 fractions were collected, and the remaining hSMUG1-containing fractions rechromatographed on MonoS as above. The pooled hSMUG1 fraction was apparently homogeneous as determined by SDS-PAGE and silver staining. The purified protein was finally concentrated by ultrafiltration and snap-frozen in liquid N2 prior to storage at −80 °C. Polyclonal PU101 against the hUNG catalytic domain was prepared as described (6Slupphaug G. Eftedal I. Kavli B. Bharati S. Helle N.M. Haug T. Levine D.W. Krokan H.E. Biochemistry. 1995; 34: 128-138Crossref PubMed Scopus (246) Google Scholar). Polyclonal PSM1 against hSMUG1 was prepared by subcutaneous injection of 66 μg of purified, recombinant hSMUG1 in Freund's complete adjuvant at the neck and flank of New Zealand White rabbits. Subsequent immunizations with the same amount of hSMUG1 in incomplete Freund's adjuvant were performed as above at 3-week intervals, and the final bleed 10 days after the last immunization. IgG-fractions of PU101 and PSM1 were purified on protein A-Sepharose HiTrap columns (AmershamBiosciences) before being used in inhibition experiments. Spontaneously transformed human keratinocytes HaCaT, colorectal carcinoma CX-1, and HeLa cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 0.03% glutamine, and 0.1 mg/ml gentamicin at 5% CO2. Cells were harvested in the logarithmic growth phase by trypsinization, washed twice in ice-cold phosphate-buffered saline prior to isolation of nuclei. Subsequently, CX-1 and HeLa were washed once in ice-cold isotonic buffer (20 mm HEPES-NaOH (pH 7.8), 1 mmMgCl2, 5 mm KCl, 1 mm DTT, and 250 mm sucrose). The pellet was resuspended in hypotonic buffer (isotonic buffer without sucrose) containing Complete®protease inhibitor mix (1 tablet/50 ml) and left on ice for 45 min to swell. Cells were lysed by 10–20 strokes in a Dounce homogenizer (tight-fitting pestle), and nuclei pelleted by centrifugation at 600 × g for 6 min. The pellets were resuspended in hypertonic buffer (hypotonic buffer with 0.5 m NaCl and 25% glycerol), and incubated at 4 °C on a rotary shaker at high speed for 30 min to extract nuclear proteins. Nuclear debris was removed by centrifugation at 12,000 × g for 15 min, protein concentration in the supernatant measured using the Bio-Rad protein assay (BSA as standard), and the nuclear extract aliquoted, snap-frozen in liquid N2, and stored at −80 °C until use. Nuclei from HaCaT keratinocytes were prepared using the NuClear® nuclear extraction kit (Sigma) according to the protocol form the manufacturer, using isotonic buffer and detergent. Serum starvation, [3H]thymidine pulse labeling, and harvesting of HaCaT cells were as described (27Haug T. Skorpen F. Aas P.A. Malm V. Skjelbred C. Krokan H.E. Nucleic Acids Res. 1998; 26: 1449-1457Crossref PubMed Scopus (92) Google Scholar). Polyclonal PSM1 antiserum was covalently coupled to magnetic protein A Dynabeads (Dynal, Norway) according to the instructions from the manufacturer. Cells (5 × 105) from each time point were resuspended in 1.5 ml of lysis buffer (1× Tris-buffered saline, 1 mg/ml BSA, 1% Triton X-100, 1 tablet/50 ml Complete® maxi+EDTA (Roche) protease inhibitors) and passed 10 times through a 21-gauge syringe needle. PSM1-coupled magnetic beads (10 μl) were added, and the extract incubated on a rotary shaker for 2 h at 4 °C. After extensive washing, the beads were eluted by boiling in SDS loading buffer and subjected to SDS-PAGE. After electroblotting to polyvinylidene difluoride membranes, bands were visualized by standard Western analysis using PSM1 as primary antibodies, HRP swine × rabbit secondary antibodies (Dako), and ECL (Amersham Biosciences). PCR was run on DNA from IMAGE clone cDNA_id 726197 encoding hSMUG1 in pT7T3D, using Pfu polymerase (Stratagene), a T7 primer, and the primer 5′-ATT TCA ACA GCA GTG GCA GC-3′. The PCR product was purified by QIAQuick PCR purification (Qiagen) and digested withEcoRI. The PCR fragment (EcoRI/blunt) encoding hSMUG1 was cloned into EcoRI/SmaI of pEGFP-N1 (Clontech). hSMUG1 and hUNG2 cDNA from pSMUG1-EGFP and pUNG2EGFP (4Otterlei M. Warbrick E. Nagelhus T.A. Haug T. Slupphaug G. Akbari M. Aas P.A. Steinsbekk K. Bakke O. Krokan H.E. EMBO J. 1999; 18: 3834-3844Crossref PubMed Scopus (295) Google Scholar) were ligated into pEYFP-N1 (Clontech) by transferring theNheI/AgeI fragment into the corresponding sites in pEYFP-N1 to make SMUG1-EYFP and UNG2-EYFP, respectively. To make the C-terminal fusion of SMUG1 with EYFP, theEcoRI/BamHI fragment from SMUG1-EYFP was ligated with EcoRI/BamHI-digested pEYFP-C1 (Clontech). The resulting vector was digested with SacI, the 3′ overhang removed by T4 DNA polymerase, and the vector re-ligated to make EYFP-SMUG1. The fusion constructs were verified to be in-frame by sequencing. The construct carrying the hUNG2 promoter, pGL2-PA (27Haug T. Skorpen F. Aas P.A. Malm V. Skjelbred C. Krokan H.E. Nucleic Acids Res. 1998; 26: 1449-1457Crossref PubMed Scopus (92) Google Scholar), was digested with HindIII/EcoRV to remove the luciferase gene. The HindIII/DraI fragment from pUNG2-EYFP was then ligated withHindIII/EcoRV-digested pGL2-PA. From this plasmid, the SacI/NotI fragment containing ProA-UNG2-EYFP was excised, and ligated intoSacI/NotI-digested pEGFP-1 (Clontech), thus replacing the EGFP-coding sequence in the vector and giving pProA-UNG2-EYFP. ECFP-PCNA fusion constructs were prepared from pEGFP-PCNAL2 (28Leonhardt H. Rahn H.P. Weinzierl P. Sporbert A. Cremer T. Zink D. Cardoso M.C. J. Cell Biol. 2000; 149: 271-280Crossref PubMed Scopus (447) Google Scholar), which contains the SV40 NLS in front of PCNA in a C-terminal fusion with EGFP. The SV40 NLS was amplified by using PCR primers containing the sites for the restriction enzymesNheI and AgeI. After digestion withNheI and AgeI, the fragment was cloned into the corresponding sites in the pECFP-C1 vectors (Clontech). Next, theBsrGI (in EGFP)/XbaI (behind PCNA) fragment from the pEGFP-PCNAL2 construct was cloned into theBrsGI/XbaI sites of pECFP-C1 vector with the SV40 NLS N-terminally, giving pNLS-ECFP-PCNA (named pECFP-PCNA). HeLa cells were transiently and stably transfected by using calcium phosphate (Profection, Promega) according to the recommendations from the manufacturer. Stable transfectants of HeLa with pProA-UNG2-EYFP were further cloned by use of limited dilutions in 96-microwell dishes in medium containing G418. The cells were examined in a Zeiss LSM 510 laser-scanning microscope equipped with a Plan-Apochromat 63×/1.4 oil immersion objective. The 458-nm laser line was used for excitation of ECFP (detected at 480 nm < λECFP < 520 nm) and the 514-nm laser line for EYFP (detected at λEYFP > 560 nm). Unless otherwise stated, UDG activity was measured in 20 μl of assay mixture containing (final) 20 mm Tris-HCl (pH 7.5), 10 mm NaCl, 1 mm EDTA, 1 mm DTT, 0.5 mg/ml bovine serum albumin, 1.8 μm [3H]dUMP-containing calf thymus DNA (specific activity 0.5 mCi/μmol) and varying amounts of enzyme. The mixture was incubated 10 min at 30 °C, and the amount of released uracil measured as described (29Krokan H. Wittwer C.U. Nucleic Acids Res. 1981; 9: 2599-2613Crossref PubMed Scopus (151) Google Scholar). Kinetic assays using 0.24–21.5 μm calf thymus substrate were performed in the buffer above. In separate experiments kinetic parameters were measured in the presence of either 7.5 mm MgCl2 or 7.5 mm MgCl2 and 0.44 ng/μl APE1 in the same buffer (molar ratio of APE1/glycosylase at least 10/1). The amount of hUNG2 or hSMUG1 used in the kinetic assays was adjusted to ensure that less than 30% of the substrate was consumed, to ensure linearity of the assay. Kinetic parameters were calculated using the Enzpack for Windows version 1.4 software package (Biosoft) using the method of Wilkinson. Enzyme kinetic parameters were also analyzed using a panel of oligonucleotide substrates. These were prepared by33P-5′-end labeling of 19-mer PAGE-purified oligonucleotides containing U or HmU at a central position (U141, 5′-CATAAAGTGUAAAGCCTGG-3′; HmU141, 5′-CATAAAGTGHmUAAAGCCTGG-3′). To generate double-stranded substrates, the labeled strands were annealed to a 50% excess of the complementary strand containing either A or G opposite U or HmU. Varying substrate concentrations (0.1–15 μm) were made by addition of identical non-labeled oligonucleotides. The kinetic assays (10 μl total) were performed in the same buffer as for the [3H]dUMP-containing calf thymus DNA in the presence of MgCl2. After 10 min of incubation at 30 °C, the reactions were stopped by addition of 50 μl of 1 m piperidine and heated at 90 °C for 20 min to cleave at AP sites. Piperidine was removed by drying under vacuum, and the oligonucleotides redissolved in 65% formamide loading buffer and analyzed by denaturing PAGE (12% polyacrylamide, 7 murea). Cleaved and uncleaved oligonucleotides were quantified by phosphorimaging. Kinetic parameters were calculated as above. In separate experiments, the activities of hUNG2 and hSMUG1 were directly compared by using ds oligonucleotides (20 nm final) having the same sequence as above, and containing U:G, HmU:G, 5-fluorouracil:A (FU:A), 3,N4-ethenocytosine:G (εC:G), or 5-hydroxyuracil:G (5-OHU:G). To detect activity against the weaker substrates, reactions contained varying amounts of hUNG2 or hSMUG1 (0–10 ng/μl final) and were incubated at 37 °C for 30 in the presence of 7.5 mm Mg2+ with or without APE1 (0.1 ng/μl final). Inhibition of hSMUG1 and hUNG2 by polyclonal antibodies or oligonucleotides containing AP sites was monitored after incubation of the purified enzymes or cell extracts with inhibitor for 10 min on ice prior to the enzyme assays. Double-stranded plasmid DNA substrates containing uracil at defined positions were prepared essentially as described (30Frosina G. Capelli E. Fortini P. Dogliotti E. Henderson D.E. Methods in Molecular Biology. Humana Press Inc., Totowa, NJ1998: 301-315Google Scholar). Briefly 20 μg of ssDNA (pGEM-3zf+) was annealed to 4.2 μg of a 22-mer complementary oligonucleotide containing uracil and synthesis of duplex DNA carried out in the presence of T4 DNA polymerase, T4 DNA ligase, and T4 gene 32 ssDNA-binding protein at 37 °C for 2h. Closed circular DNA duplex molecules were purified by CsCl gradient centrifugation. The base excision repair mixtures (50 μl) contained (final) 40 mm HEPES-KOH (pH 7.8), 70 mm KCl, 5 mm MgCl2, 0.5 mm DTT, 2 mm ATP, 20 μm dATP, 20 μm dGTP, 8 μm dTTP or dCTP depending on the isotope used, 8 μm phosphocreatine, 0.36 mg/ml BSA, 1 μg/ml

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