cDNA cloning, sequence analysis and expression of a mouse 44-kDa nuclear protein copurified with DNA repair factors for acid-depurinated DNA.
We purified a 44-kDa nuclear protein from salt-extract of permeable mouse ascites sarcoma cells in an effort to isolate factors involved in the repair of acid-depurinated DNA. It was copurified with a major AP endonuclease (APEX nuclease) by sequential column chromatography then further purified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis as a possible DNA repair support factor. Its partial amino acid sequences were determined, and a cDNA clone for the protein was isolated from a mouse T-cell cDNA library using long degenerate oligonucleotide probes deduced from the amino acid sequence. The complete nucleotide sequence of the cDNA (1.7 kilobases) was determined. Northern hybridization using this cDNA detected two transcripts: 1.8 kb being the major one and 2.6 kb being the minor one. The complete amino acid sequence for the protein predicted from the nucleotide sequence of the cDNA indicates that the 44-kDa protein consists of 394 amino acids with a calculated molecular weight of 43,698. In tests performed thus far, the recombinant 44-kDa protein expressed in Escherichia coli has not expressed any repair-support activity. It remains to be analyzed whether the protein attains this activity after appropriate posttranslational modifications. Most parts of the 44-kDa protein cDNA and the deduced amino acid sequence were found to be identical to those of the protein p38-2G4, recently reported as a cell cycle-specifically modulated nuclear protein of 38kDa. The p38-2G4 may be a truncated form of the present 44-kDa protein.
Nakagawa
Y
Y
Department of Molecuclar Biology, Okayama University Medical School, Japan.
Watanabe
S
S
Akiyama
K
K
Sarker
A H
AH
Tsutsui
K
K
Inoue
H
H
Seki
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004260:DNA Repair; D018076:DNA, Complementary / Q000737:chemistry; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D029968:Escherichia coli Proteins; D008190:Lyases / Q000302:isolation & purification*; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis
D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase