Cell-cycle regulation, intracellular sorting and induced overexpression of the human NTH1 DNA glycosylase involved in removal of formamidopyrimidine residues from DNA.
Endonuclease III (Nth) of Escherichia coli is a DNA glycosylase essential for the removal of oxidised pyrimidine base residues from DNA. Several eukaryotic homologues have recently been identified and shown to have biochemical properties similar to those of Nth. However, some of the eukaryotic counterparts also appear to remove imidazole ring-opened purine residues (faPy), a property not shared by the enzymes of bacterial origin. Here, we show that the human enzyme also possesses efficient faPy DNA glycosylase activity as indicated both from studies of the purified protein and induced overexpression of the human NTH1 cDNA in HeLa cells. We constructed green fluorescent protein-tagged hNTH1 fusion proteins to study the cellular localisation of hNTH1 and found strong and exclusive sorting to the nucleus. Studies with synchronised cells showed that the expression of hNTH1 is regulated during the cell cycle with increased transcription during early and mid S-phase.
Luna
L
L
Department of Molecular Biology, Institute of Medical Microbiology, University of Oslo, The National Hospital, N-0027, Oslo, Norway. luisa.luna@labmed.uio.no
Bjørås
M
M
Hoff
E
E
Rognes
T
T
Seeberg
E
E
D000595:Amino Acid Sequence; D001692:Biological Transport; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D004260:DNA Repair / Q000235:genetics*; D043382:DNA-Formamidopyrimidine Glycosylase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004318:Doxycycline / Q000494:pharmacology; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes / Q000201:enzymology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000235:genetics; D011743:Pyrimidines / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016196:S Phase / Q000235:genetics
D029968:Escherichia coli Proteins; D019913:Nuclear Localization Signals; D011743:Pyrimidines; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; C468347:NTHL1 protein, human; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D004318:Doxycycline