P53-associated 3'-->5' exonuclease activity in nuclear and cytoplasmic compartments of cells.

Abstract

The tumor suppressor protein p53 plays an important role in maintenance of the genomic integrity of cells. p53 possesses an intrinsic 3'-->5' exonuclease activity. p53 was found in the nucleus and in the cytoplasm of the cell. In order to evaluate the subcellular location and extent of p53-associated 3'--> 5' exonuclease activity, we established an in vitro experimental system of cell lines with different nuclear/cytoplasmic distribution of p53. Nuclear and cytoplasmic extracts obtained from LCC2 cells (expressing a high level of cytoplasmic wild-type p53), MCF-7 cells (expressing a high level of wild-type nuclear p53), MDA cells (expressing mutant p53) and H1299 cells (p53-null) were subjected to the analysis of exonuclease activity. Interestingly, 3'-->5' exonuclease was predominantly cytoplasmic; the nuclear extracts derived from all cell lines tested, exerted a low level of exonuclease activity. Cytoplasmic extracts of LCC2 cells, with a high level of wild-type p53, showed an enhanced exonuclease activity in comparison to those expressing either a low level of wild-type p53 (in MCF-7 cells) or the mutant p53 (in MDA cells). Evidence that exonuclease function detected in cytoplasmic extracts is attributed to the p53 is supported by several facts: First, this activity closely parallels with levels and status of endogenous cytoplasmic p53. Second, immunoprecipitation of p53 from cytoplasmic extracts of LCC2 cells markedly reduced the exonuclease activity. Third, the observed 3'-->5' exonuclease in cytoplasmic fraction of LCC2 cells displays identical biochemical properties characteristic of recombinant wild-type p53. The biochemical functions include: (a) substrate specificity; exonuclease hydrolyzes single-stranded DNA in preference to double-stranded DNA and RNA/DNA template-primers, (b) efficient excision of 3'-terminal mispairs from DNA/DNA and RNA/DNA substrates, (c) the preferential excision of purine-purine mispairs over purine-pyrimidine mispairs and (d) functional interaction with exonuclease-deficient DNA polymerase, for example, murine leukemia virus reverse transcriptase (representing a relatively low fidelity enzyme), thus enhancing the fidelity of DNA synthesis by excision of mismatched nucleotides from the nascent DNA strand. Taken together, the data demonstrate that wild-type p53 in cytoplasm, in its noninduced state, is functional; it displays intrinsic 3'-->5' exonuclease activity. The possible role of p53-associated 3'-->5' exonuclease activity in DNA repair in nucleus and cytoplasm is discussed.