KsgA, a 16S rRNA adenine methyltransferase, has a novel DNA glycosylase/AP lyase activity to prevent mutations in Escherichia coli.
The 5-formyluracil (5-foU), a major mutagenic oxidative damage of thymine, is removed from DNA by Nth, Nei and MutM in Escherichia coli. However, DNA polymerases can also replicate past the 5-foU by incorporating C and G opposite the lesion, although the mechanism of correction of the incorporated bases is still unknown. In this study, using a borohydride-trapping assay, we identified a protein trapped by a 5-foU/C-containing oligonucleotide in an extract from E. coli mutM nth nei mutant. The protein was subsequently purified from the E. coli mutM nth nei mutant and was identified as KsgA, a 16S rRNA adenine methyltransferase. Recombinant KsgA also formed the trapped complex with 5-foU/C- and thymine glycol (Tg)/C-containing oligonucleotides. Furthermore, KsgA excised C opposite 5-foU, Tg and 5-hydroxymethyluracil (5-hmU) from duplex oligonucleotides via a beta-elimination reaction, whereas it could not remove the damaged base. In contrast, KsgA did not remove C opposite normal bases, 7,8-dihydro-8-oxoguanine and 2-hydroxyadenine. Finally, the introduction of the ksgA mutation increased spontaneous mutations in E. coli mutM mutY and nth nei mutants. These results demonstrate that KsgA has a novel DNA glycosylase/AP lyase activity for C mispaired with oxidized T that prevents the formation of mutations, which is in addition to its known rRNA adenine methyltransferase activity essential for ribosome biogenesis.
Zhang-Akiyama
Qiu-Mei
QM
Laboratory of Radiation Biology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan. qmzhang@kingyo.zool.kyoto-u.ac.jp
Morinaga
Hironobu
H
Kikuchi
Masahiro
M
Yonekura
Shin-Ichiro
S
Sugiyama
Hiroshi
H
Yamamoto
Kazuo
K
Yonei
Shuji
S
D000595:Amino Acid Sequence; D045647:DNA Glycosylases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry / Q000235:genetics; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D029968:Escherichia coli Proteins; D008780:Methyltransferases; C047346:16S rRNA (adenine(1518)-N(6)-adenine(1519)-N(6))-dimethyltransferase; D043224:Deoxyribonuclease (Pyrimidine Dimer); C468346:Nei protein, E coli; D045647:DNA Glycosylases; C059701:cytosine-DNA glycosidase; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase