Valerie K, Green A P, de Riel J K, Henderson E E
Cancer Res. 1987 Jun 1;47(11):2967-71.
In this paper we report both transient and stable complementation of pyrimidine dimer repair in xeroderma pigmentosum cells by the denV gene of bacteriophage T4, coding for endonuclease V, a dimer-specific DNA glycosylase. Cotransfection with pRSVdenV in SV40-transformed XP12RO(M1) cells (complementation group A) restored transient expression of an indicator plasmid (pRSVcat) bearing a UV-inactivated chloramphenicol acetyltransferase (cat) gene. In addition, XP12RO(M1) clones stably transformed by pRSVdenV-SVgpt expressed transient chloramphenicol acetyltransferase activity when transfected with UV-inactivated pRSVcat plasmid. These clones also showed partial restoration of colony forming ability and excision repair synthesis after UV irradiation. Immunofluorescence, using an endonuclease V polyclonal antibody, showed the presence of the phage glycosylase in stably transformed xeroderma pigmentosum cells. The cotransfection assay affords a rapid, sensitive procedure to screen for functional cloned DNA repair genes and to test mutant cells for the deficiency of specific steps in DNA repair, such as incision.
在本文中，我们报道了噬菌体T4的denV基因对着色性干皮病细胞嘧啶二聚体修复的瞬时和稳定互补作用，该基因编码内切核酸酶V，一种二聚体特异性DNA糖基化酶。在SV40转化的XP12RO(M1)细胞（互补组A）中用pRSVdenV共转染，恢复了携带紫外线灭活的氯霉素乙酰转移酶（cat）基因的指示质粒（pRSVcat）的瞬时表达。此外，用紫外线灭活的pRSVcat质粒转染时，由pRSVdenV-SVgpt稳定转化的XP12RO(M1)克隆表达瞬时氯霉素乙酰转移酶活性。这些克隆在紫外线照射后还显示出集落形成能力和切除修复合成的部分恢复。使用内切核酸酶V多克隆抗体的免疫荧光显示，在稳定转化着色性干皮病细胞中存在噬菌体糖基化酶。共转染测定提供了一种快速、灵敏的方法，用于筛选功能性克隆的DNA修复基因，并检测突变细胞在DNA修复中特定步骤（如切口）的缺陷。
