Identification of a point mutation in the cDNA of the catalytic subunit of DNA polymerase alpha from a temperature-sensitive mouse FM3A cell line.

Abstract

DNA polymerase alpha is a key enzyme in eukaryotic chromosomal DNA replication. tsFT20 is a temperature-sensitive mutant cell line derived from mouse mammary carcinoma FM3A cells, and the cells contain heat-labile DNA polymerase alpha and are arrested at the S phase at the nonpermissive temperature. We isolated cDNA of the catalytic subunit of DNA polymerase alpha from tsFT20 cells. DNA sequence analysis revealed that the cDNA from tsFT20 has a single mutation, a cytosine to thymine substitution that changes amino acid 1180 from serine to phenylalanine. We have also shown that tsFT20 cells could be rescued by transfection with the wild-type cDNA. These results demonstrate that the point mutation in the gene of DNA polymerase alpha causes the temperature-sensitive phenotype of tsFT20 cells and provide additional evidence that DNA polymerase alpha is essential for chromosomal replication in mammalian cells. We also detected mutation sites in one spontaneous and six N-methyl-N'-nitro-N-nitrosoguanidine-induced growth revertants of tsFT20 cells by single strand conformation polymorphism analyses and direct sequencing. All revertant cell lines had a second point mutation adjacent to the first mutation site in tsFT20 cells.