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 mo use 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 electro phoresis as a possible DNA repair support factor. Its partial amino ac id sequences were determined, and a cDNA clone for the protein was iso lated from a mouse T-cell cDNA library using long degenerate oligonucl eotide probes deduced from the amino acid sequence. The complete nucle otide sequence of the cDNA (1.7 kilobases) was determined. Northern hy bridization 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 seq uence for the protein predicted from the nucleotide sequence of the cD NA 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 e xpressed any repair-support activity. It remains to be analyzed whethe r the protein attains this activity after appropriate posttranslationa l modifications. Most parts of the 44-kDa protein cDNA and the deduced amino acid sequence were found to be identical to those of the protei n p38-2G4, recently reported as a cell cycle-specifically modulated nu clear protein of 38 kDa. The p38-2G4 may be a truncated form of the pr esent 44-kDa protein.