Nuclear localization of the human mutY homologue hMYH.
The cDNA of the human mutY homologue (hMYH) was cloned from the total RNA of the tumor cell line SU-DHL-1 by reverse transcription-polymerase chain reaction (RT-PCR). Expression of hMYH in a plasmid can partially revert the mutator phenotype of the Escherichia coli mutY mutant MK609(DE3). The majority of the recombinant hMYH protein in E. coli was precipitated in the inclusion bodies. A minor fraction of the soluble recombinant protein was concentrated as the source of the protein in the activity assay. Recombinant hMYH displayed both glycosylase and AP lyase activity. Three independent rabbit antisera against an N-terminal peptide, HY90, a recombinant C-terminal fragment, and the full-length hMYH recombinant protein were prepared and affinity-purified, and these antisera recognized the 59 kDa endogenous hMYH protein in HeLa cells. Immunofluorescent staining experiments with these three antisera showed a consistent nuclear distribution of hMYH, excluding the nucleoli. This nuclear staining pattern was abolished if the antisera were incubated with specific peptide/protein competitors, whereas the staining pattern was unaffected if the antisera were incubated with nonspecific peptide competitors. Consistent with the immunofluorescent staining results, a flag-tagged transfected hMYH also showed a nuclear staining pattern excluding the nucleoli. These results suggest that hMYH is indeed a functional homologue of E. coli MutY and is localized in the nuclei of mammalian cells.
Tsai-Wu
J J
JJ
Department of Clinical Research, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China.
Su
H T
HT
Wu
Y L
YL
Hsu
S M
SM
Wu
C H
CH
D000595:Amino Acid Sequence; D020137:Base Pair Mismatch; D015153:Blotting, Western; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004260:DNA Repair; D018076:DNA, Complementary / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D029968:Escherichia coli Proteins; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis* / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase