Nucleic Acids Research, 2003, Vol. 31, No. 8 2045±2055 DOI: 10.1093/nar/gkg319 A novel uracil-DNA glycosylase family related to the helix±hairpin±helix DNA glycosylase superfamily Ji Hyung Chung1,2,3, Eun Kyoung Im2,3, Hyun-Young Park1,2,3, Jun Hye Kwon2,4, Seahyoung Lee2, Jaewon Oh2, Ki-Chul Hwang2,3, Jong Ho Lee1,2,3 and Yangsoo Jang1,2,3,4,* 1Yonsei Research Institute of Aging Science, 2Cardiovascular Research Institute, 3Cardiovascular Genome Center and 4Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Yonsei University, Seoul, 120-752, Korea Received January 23, 2003; Revised and Accepted February 21, 2003 ABSTRACT catalyze the major repair process, the base excision repair (BER) pathway, which is initiated by removal of the damaged Cytosine bases can be deaminated spontaneously base (2,3). Among those DNA glycosylases, uracil-DNA to uracil, causing DNA damage. Uracil-DNA glyco- glycosylase (UDG) was the ®rst to be discovered (4), and it sylase (UDG), a ubiquitous uracil-excising enzyme acts as a major repair enzyme that protects DNA from found in bacteria and eukaryotes, is one of the mutational damages caused by the misincorporation of uracil enzymes that repair this kind of DNA damage. To due to a polymerase error or deamination of cytosine. This date, no UDG-coding gene has been identi®ed in repair enzyme hydrolyzed the N-glycosidic bond. As a result, Methanococcus jannaschii, although its entire uracil base is released from the DNA backbone, leaving an genome was deciphered. Here, we have identi®ed abasic site behind. This uracil-removing repair mechanism and characterized a novel UDG from M.jannaschii seems to be one of the essential and common DNA designated as MjUDG. It ef®ciently removed uracil repair systems, since UDG-homologous genes and their from both single- and double-stranded DNA. MjUDG corresponding enzymes can be found in a variety of organisms also catalyzes the excision of 8-oxoguanine from (2,3). DNA. MjUDG has a helix±hairpin±helix motif and a The genes that encode uracil-excising enzyme have been found in bacteria (4), viruses (5) and eukaryotes, including [4Fe±4S]-binding cluster that is considered to be human (6,7). They can be generally classi®ed into six families important for the DNA binding and catalytic activity. [®ve UDG families plus the mismatch-speci®c DNA glycosyl- Although MjUDG shares these features with other ase (MIG) family] according to their differences in substrate structural families such as endonuclease III and recognition and amino acid sequence (8±10). The six families mismatch-speci®c DNA glycosylase (MIG), unique are the Ung family (family I) (4,11,12), the MUG/TDG family conserved amino acids and substrate speci®city (family II) (13,14), the sMUG family (family III) (15), the distinguish MjUDG from other families. Also, a thermostable UDG family (family IV, TmUDG) (16±18), homologous member of MjUDG was identi®ed in the UDG-B family (family V) (9,19) and the MIG family. Aquifex aeolicus. We report that MjUDG belongs to However, these ®ve different UDG families show limited a novel UDG family that has not been described sequence similarity, although they share two active site motifs to date. (motifs A and B) (8,9). Genes analogous to UDG also appear to be present in a variety of thermophilic eubacteria and archaea. Several INTRODUCTION thermostable UDGs (thermostable UDG family, family IV) Damaged DNA lesions occur when DNA is modi®ed by UV have been characterized from genomes of thermophiles such light, ionizing radiation, reactive oxygen species or chemical as Thermotoga maritima (16), Archaeoglobus fulgidus (17) mutagens. Uracil residues can also be produced in DNA via and Pyrobaculum aerophilum (18) since UDG activities were the spontaneous deamination of existing cytosines or the detected in the hyperthermophile extracts (20). These proteins misincorporation of dUMP during DNA synthesis. Uracil is are capable of removing uracil from uracil-mismatched duplex biologically important because cytosine to thymine transition substrates and are also active on single-stranded DNA is induced at the site of cytosine deamination in DNA (1). containing uracil. Motif A of this family does not contain However, most organisms have repair enzymes that are aspartic acid or asparagine at the corresponding position of the speci®c for this deaminated base in DNA. DNA glycosylases catalytic residue in motif A of other families. However, motif *To whom correspondence should be addressed at Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul, 120-752, Korea. Tel: +82 2 361 7266; Fax: +82 2 365 1878; Email: [email protected] The authors wish it to be known that, in their opinion, the ®rst two authors should be considered as joint First Authors Nucleic Acids Research, Vol. 31 No. 8 ã Oxford University Press 2003; all rights reserved 2046 Nucleic Acids Research, 2003, Vol. 31, No. 8 A (-GEAPG-) of this family has glutamic acid, which may generated by insertion of the PCR product into the cloning participate in the activation of the catalytic water molecule (8). site (NdeI and HindIII) of pET28a in order to express MjUDG Relatively recently, another hyperthermophilic UDG family, protein with a polyhistidine tag. The nucleotide sequence was family V (UDG-B), has been identi®ed in P.aerophilum (9) determined by the dideoxy chain termination method as and Thermus thermophilus (19). The proteins belonging to this described in the instruction manual of the Perkin-Elmer cycle family have a prominent structural feature that is the absence sequencing kit (Perkin-Elmer, MA). Gene-speci®c primers of of any polar amino acid residues within motif A (-GLAPA/G-). MJ1434 were used to con®rm the correct insertion. Brie¯y, the One of the members of the hyperthermophilic archaea is sequencing reaction products were separated by 6% acryl- Methanococcus jannaschii that grows optimally at pressures amide gel electrophoresis, and the sequence was determined of up to >200 atm and 85°C. Although the entire genome using an ABI 373 automatic DNA sequencer (Applied sequence has now been determined, making it the ®rst archael Biosystems, CA). organism to be sequenced completely (21), the UDG-encoding gene of M.jannaschii has not yet been identi®ed. Hyper- Protein puri®cation thermophilic organisms living in a high temperature environ- The recombinant plasmid pET28a-MjUDG was introduced ment are at especially high risk of DNA damage by cytosine into E.coli strain BL21 (DE3). Then the E.coli BL21 (DE3) deamination because high temperature can signi®cantly harboring pET28a-MjUDG were inoculated into LB medium promote base deamination (22). Thus it is plausible that they (1% tryptone, 0.5% yeast extract and 0.5% NaCl) containing may have more effective DNA damage repair systems 50 mg/ml of kanamycin to a density of A600 1.0 at 37°C. compared with other organisms. In this study, we identi®ed Recombinant proteins were induced with 0.5 mM isopropyl-b- and characterized, to our knowledge for the ®rst time, a UDG D-thiogalactopyranoside (IPTG) at 30°C for 6 h. The cells from M.jannaschii (MjUDG). MjUDG was cloned from the were centrifuged at 6000 r.p.m. for 20 min, and then cell open reading frame (ORF) MJ1434 encoding a protein of 220 pellets were resuspended in buffer A (50 mM NaH2PO4, amino acids with a mol. wt of 26 kDa. Our results show that 300 mM NaCl pH 8.0). Cells were disrupted by ultrasonication the MjUDG catalyzes the removal of uracil from both single- and the lysates were centrifuged at 15 000 r.p.m. for 30 min. and double-stranded DNA. In addition, they also revealed that The supernatant was applied to an Ni-NTA±agarose resin MjUDG has an 8-oxoguanine (8-oxoG)-excising function that column (Qiagen) pre-equilibrated in buffer A at a ¯ow rate of has not been found in other UDG family enzymes. Based on 1 ml/min. The ¯ow-through was discarded and proteins were these observations and the result of extended sequence washed 10 times with additional column volumes of buffer A. analysis of known DNA glycosylases, we conclude that MjUDG was eluted from the column with buffer B (50 mM MjUDG belongs to a novel family that has not been identi®ed NaH2PO4, 300 mM NaCl, 300 mM imidazole pH 8.0). The to date. Therefore, we named it the MjUDG family (family VI). protein fractions were identi®ed by 15% SDS±PAGE. The volume of sample fractions was reduced to 20 ml by concentration with an Amicon YM10 membrane (Millipore, MATERIALS AND METHODS MA), then cleaved using thrombin protease (10 U/mg of fusion protein). The cleavage mixture of fusion protein was Bacterial strains and reagents dialyzed in buffer A and applied to a Superdex-75 gel ®ltration The expression strain BL21 (DE3) and plasmid pET28a with a FPLC column. The fractions containing homogeneous polyhistidine tag were obtained from Novagen (WI). The MjUDG were collected using a fraction collector and hyperthermophilic archaeon M.jannaschii (DSM 2661) was concentrated by ultra®ltration using an Amicon YM10 purchased from the Deutsche Sammlung von Mikroorganism membrane. The pure MjUDG protein obtained was stored at und Zellkultren GmbH (DSM, Braunschweig, Germany). ±80°C at a concentration of 1 mg/ml. Methanococcus jannaschii was cultivated under conditions as DNA substrates reported previously (23). Genomic DNA was prepared using a Qiagen Genomic DNA Midi Kit (Qiagen GmbH, Hilden, The oligonucleotide sequences used in this work were 32mer Germany). Restriction endonuclease, T4 DNA ligase, strands (5¢-GGATCCTCTAGAGTCXACCTGCAGGCATG- Escherichia coli endonuclease III (EndoIII) and UDG were CAA-3¢) or 39mer strands (5¢-GGATCCTCTAGAGTCY- purchased from New England Biolabs (MA).