YEAST OPEN READING FRAME YCR14C ENCODES A DNA BETA-POLYMERASE-LIKE ENZYME

We have shown by activity gel that overexpression in E.coli of a yeast chromosome 3 open reading frame (ORF) designated YCR14C and bearing h omology to mammalian DNA polymerases beta results in a new DNA polymer ase in the host cells. The molecular mass of this enzyme corresponded to the YCR14C-predicted 67 kDa protein, and NH2-terminal amino acid se quencing confirmed that the expressed protein was encoded by the yeast ORF. This new yeast DNA polymerase was purified to homogeneity from E .coli. In a fashion similar to that of mammalian beta-polymerases, the purified yeast enzyme exhibited distributive DNA synthesis on DNA sub strate with a single-stranded template and processive gap-filling synt hesis on a short-gapped DNA substrate. Activity of this yeast beta-pol ymerase-like enzyme was sensitive to the beta-polymerase inhibitor ddN TP and resistant to both 1 mM NEM and neutralizing antibody to E.coli DNA polymerase 1. These results, therefore, indicate that YCR14C encod es a DNA beta-polymerase-like enzyme in yeast, and we name it DNA poly merase IV. Yeast strains harboring a deletion mutation of the pol IV g ene are viable, they exhibit no increase in sensitivity to ultraviolet light, ionizing radiation or alkylating agents, and sporulation and s pore viability are not affected in the mutant.