Prasad R, Widen S G, Singhal R K, Watkins J, Prakash L, Wilson S H
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-0851.
Nucleic Acids Res. 1993 Nov 25;21(23):5301-7. doi: 10.1093/nar/21.23.5301.
We have shown by activity gel that overexpression in E. coli of a yeast chromosome 3 open reading frame (ORF) designated YCR14C and bearing homology to mammalian DNA polymerases beta results in a new DNA polymerase in the host cells. The molecular mass of this enzyme corresponded to the YCR14C-predicted 67 kDa protein, and NH2-terminal amino acid sequencing 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 substrate with a single-stranded template and processive gap-filling synthesis on a short-gapped DNA substrate. Activity of this yeast beta-polymerase-like enzyme was sensitive to the beta-polymerase inhibitor ddNTP and resistant to both 1 mM NEM and neutralizing antibody to E. coli DNA polymerase I. These results, therefore, indicate that YCR14C encodes a DNA beta-polymerase-like enzyme in yeast, and we name it DNA polymerase IV. Yeast strains harboring a deletion mutation of the pol IV gene are viable, they exhibit no increase in sensitivity to ultraviolet light, ionizing radiation or alkylating agents, and sporulation and spore viability are not affected in the mutant.
我们通过活性凝胶证明，在大肠杆菌中过表达一个名为YCR14C的酵母3号染色体开放阅读框（ORF），该阅读框与哺乳动物DNA聚合酶β具有同源性，结果在宿主细胞中产生了一种新的DNA聚合酶。这种酶的分子量与YCR14C预测的67 kDa蛋白相对应，并且氨基末端氨基酸测序证实表达的蛋白由酵母ORF编码。这种新的酵母DNA聚合酶从大肠杆菌中纯化至同质。与哺乳动物β-聚合酶类似，纯化的酵母酶在具有单链模板的DNA底物上表现出分布性DNA合成，在短缺口DNA底物上表现出持续性缺口填充合成。这种酵母β-聚合酶样酶的活性对β-聚合酶抑制剂ddNTP敏感，对1 mM NEM和抗大肠杆菌DNA聚合酶I的中和抗体均有抗性。因此，这些结果表明YCR14C在酵母中编码一种DNAβ-聚合酶样酶，我们将其命名为DNA聚合酶IV。携带pol IV基因缺失突变的酵母菌株是有活力的，它们对紫外线、电离辐射或烷化剂的敏感性没有增加，并且突变体中的孢子形成和孢子活力不受影响。
