Structure and activity of a thermostable thymine-DNA glycosylase: evidence for base twisting to remove mismatched normal DNA bases.
The repair of T:G mismatches in DNA is key for maintaining bacterial restriction/modification systems and gene silencing in higher eukaryotes. T:G mismatch repair can be initiated by a specific mismatch glycosylase (MIG) that is homologous to the helix-hairpin-helix (HhH) DNA repair enzymes. Here, we present a 2.0 A resolution crystal structure and complementary mutagenesis results for this thermophilic HhH MIG enzyme. The results suggest that MIG distorts the target thymine nucleotide by twisting the thymine base approximately 90 degrees away from its normal anti position within DNA. We propose that functionally significant differences exist in DNA repair enzyme extrahelical nucleotide binding and catalysis that are characteristic of whether the target base is damaged or is a normal base within a mispair. These results explain why pure HhH DNA glycosylases and combined glycosylase/AP lyases cannot be interconverted by simply altering their functional group chemistry, and how broad-specificity DNA glycosylase enzymes may weaken the glycosylic linkage to allow a variety of damaged DNA bases to be excised.
Mol
Clifford D
CD
Department of Molecular Biology MB4, Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Rd, La Jolla, CA 92037, USA.
Arvai
Andrew S
AS
Begley
Thomas J
TJ
Cunningham
Richard P
RP
Tainer
John A
JA
D000595:Amino Acid Sequence; D020137:Base Pair Mismatch / Q000235:genetics*; D001665:Binding Sites; D019757:Carbon-Oxygen Lyases / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017013:Methanobacteriaceae / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013941:Thymine / Q000378:metabolism
D004268:DNA-Binding Proteins; D009711:Nucleotides; D004247:DNA; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D013941:Thymine