Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149.
Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149.
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感染突变体 H5ts149 的细胞中腺病毒 DNA 聚合酶发生改变的证据。
DOI：
10.1073/pnas.80.6.1589
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发表时间：
1983
影响因子：
11.1
通讯作者：
Horwitz,MS
中科院分区：
文献类型：
--
作者：
Friefeld,BR;Lichy,JH;Hurwitz,J;Horwitz,MS
腺病毒（Ad）血清5型突变体对体内病毒DNA合成具有温度敏感性，利用N互补组研究了一种14万道尔顿的DNA聚合酶（Pol）与8万道尔顿末端蛋白前体（pTP）共融。在非允许温度下，用N组突变体H5ts149感染HeLa细胞制备的提取物不能合成病毒DNA。通过添加从野生型ad感染细胞质中纯化的Pol，这些提取物中的缺陷被特异性地逆转。添加不含Pol的pTP不能恢复H5ts149提取物的复制活性。所研究的反应依赖于DNA模板的存在，包括起始反应（dCMP与pTP的共价附着）和含有起源序列的Ad DNA限制性内切酶片段的选择性复制。甘油梯度沉降表明，与野生型提取物相比，H5ts149提取物中代表pTP-Pol复合物的复制活性大大降低，表明突变体发生了一些改变。在野生型和h5ts149感染细胞的提取物中，在这些梯度上检测到不含Pol的pTP池。此外，在允许温度下培养的细胞制备的H5ts149提取物催化的Ad DNA的起始和延伸比野生型病毒感染的细胞制备的提取物更不容易被尿素灭活。这些结果，与H5ts149突变在一个大约足够大的开放阅读框内的图谱一起考虑，以编码140,000道尔顿DNA聚合酶[Gingeras， t.r., Sciaky， D, Gelinas， R. E., Bing-Dong， J., Yen， C. E, Kelly， M. M., Bullock， P. A, Parsons， B. L， O'Neill， K. E. & Roberts， R. J.(1982)]。医学杂志。Chem.257, 13475 - 13491;Alestrom， P., Akusjarui， G., Pettersson， M. & Pettersson， U. (1982)J。医学杂志。化学，257,13492-13498]，表明Pol是一种病毒编码蛋白，pTP也是。
The N complementation group of adenovirus (Ad) serotype 5 mutants, which are temperature sensitive for viral DNA synthesisin vivo, has been used to study a 140,000-dalton DNA polymerase (Pol) that copurified with the 80,000-dalton terminal protein precursor (pTP). Extracts prepared from HeLa cells infected with the N group mutant H5ts149 at nonpermissive temperature were unable to synthesize viral DNA. The defect in these extracts was specifically reversed by addition of the Pol purified from wild-type Ad-infected cytosol. Addition of the pTP, free of the Pol, did not restore replicative activity to H5ts149 extracts. The reactions studied depend on the presence of the DNA template and include the initiation reaction (the covalent attachment of dCMP to the pTP) and the selective replication of Ad DNA restriction endonuclease fragments containing the origin sequences. Glycerol gradient sedimentation showed that a replicative activity representing the pTP-Pol complex was greatly reduced in H5ts149 extracts as compared with wild-type extracts, suggesting some alteration in the mutant. A pool of pTP free of Pol was detected on these gradients in extracts from both wild-type and H5ts149-infected cells. In addition, the initiation and elongation of Ad DNA catalyzed by H5ts149 extracts prepared from cells grown at permissive temperatures was more labile to urea inactivation than extracts prepared from cells infected with wild-type virus. These results, considered together with the mapping of the H5ts149 mutation within an open reading frame approximately large enough to code for the 140,000-dalton DNA polymerase [Gingeras, T. R., Sciaky, D., Gelinas, R. E., Bing-Dong, J., Yen, C. E., Kelly, M. M., Bullock, P. A., Parsons, B. L., O'Neill, K. E. & Roberts, R. J. (1982)J. Biol. Chem.257, 13475-13491; Alestrom, P., Akusjarui, G., Pettersson, M. & Pettersson, U. (1982)J. Biol. Chem.257, 13492-13498], suggest that the Pol is a virally encoded protein, as is the pTP.