Nishida C, Reinhard P, Linn S
Department of Biochemistry, University of California, Berkeley 94720.
J Biol Chem. 1988 Jan 5;263(1):501-10.
When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone. This result suggests that cytosol-depleted permeabilized DNA repair-defective human fibroblasts and HeLa DNA polymerase delta might be exploited to provide a functional assay for purifying active DNA repair factors from DNA repair-proficient cells without a preknowledge of their function.
当用Brij - 58使经紫外线照射的培养二倍体人成纤维细胞透化，然后通过离心与可溶性物质分离时，从经类似处理的HeLa细胞的上清液中获得的一种可溶性因子可恢复保守的DNA修复合成。对该因子进行广泛纯化后得到一种10.2 S、220,000道尔顿的多肽，其具有DNA聚合酶以及已报道的DNA聚合酶δ II（克鲁特，J. J.，瓦尔，A. F.，和班巴拉，R. A.（1986年）《生物化学》25，26 - 36）的3'至5'核酸外切酶活性。针对KB细胞DNA聚合酶α的单克隆抗体，虽然能与HeLa DNA聚合酶α结合，但不与HeLa DNA聚合酶δ结合。此外，在微摩尔浓度下，N2 -（对正丁基苯基）- 2'-脱氧鸟苷5'-三磷酸（BuPdGTP）和2 -（对正丁基苯胺基）- 2'-脱氧腺苷5'-三磷酸（BuAdATP）是DNA聚合酶α的有效抑制剂，但不抑制DNA聚合酶δ。纯化的DNA聚合酶α和β都不能促进透化细胞中的修复DNA合成。此外，在将纯化的DNA聚合酶α抑制超过90%的条件下，针对DNA聚合酶α的单克隆抗体、BuPdGTP或BuAdATP都不能显著抑制由DNA聚合酶δ介导的DNA修复合成。因此，似乎紫外线照射诱导的DNA修复合成的主要部分可能由DNA聚合酶δ催化。当使用着色性干皮病的人二倍体成纤维细胞时，通过添加T4内切核酸酶V和DNA聚合酶δ可恢复依赖紫外线的DNA修复合成，但单独添加其中任何一种都不行。这一结果表明，可利用细胞质耗尽的透化DNA修复缺陷型人成纤维细胞和HeLa DNA聚合酶δ来提供一种功能测定方法，以便从DNA修复 proficient细胞中纯化活性DNA修复因子，而无需预先了解它们的功能。 （注：“proficient”疑为“ proficient”，推测可能是“ proficient”，表示“熟练的、精通的”，这里结合语境可能是指“DNA修复能力正常的”，但原文拼写有误，翻译时保留原文拼写并注明疑问。）
