Keim C A, Mosbaugh D W
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331.
Biochemistry. 1991 Nov 19;30(46):11109-18. doi: 10.1021/bi00110a013.
Spinach chloroplast DNA polymerase was shown to copurify with a 3' to 5' exonuclease activity during DEAE-cellulose, hydroxylapatite, and heparin-agarose column chromatography. In addition, both activities comigrated during nondenaturing polyacrylamide gel electrophoresis and cosedimented through a glycerol gradient with an apparent molecular weight of 105,000. However, two forms of exonuclease activity were detected following velocity sedimentation analysis. Form I constituted approximately 35% of the exonuclease activity and was associated with the DNA polymerase, whereas the remaining activity (form II) was free of DNA polymerase and exhibited a molecular weight of approximately 26,500. Resedimentation of form I exonuclease generated both DNA polymerase associated and DNA polymerase unassociated forms of the exonuclease, suggesting that polymerase/exonuclease dissociation occurred. The exonuclease activity (form I) was somewhat resistant to inhibition by N-ethylmaleimide, whereas the DNA polymerase activity was extremely sensitive. Using in situ detection following SDS-polyacrylamide activity gel electrophoresis, both form I and II exonucleases were shown to reside in a similar, if not identical, polypeptide of approximately 20,000 molecular weight. Both form I and II exonucleases were equally inhibited by NaCl and required 7.5 mM MgCl2 for optimal activity. The 3' to 5' exonuclease excised deoxyribonucleoside 5'-monophosphates from both 3'-terminally matched and 3'-terminally mismatched primer termini. In general, the exonuclease preferred to hydrolyze mismatched 3'-terminal nucleotides as determined from the Vmax/Km ratios for all 16 possible combinations of matched and mismatched terminal base pairs. These results suggest that the 3' to 5' exonuclease may be involved in proofreading errors made by chloroplast DNA polymerase.
菠菜叶绿体DNA聚合酶在通过二乙氨基乙基纤维素、羟基磷灰石和肝素琼脂糖柱层析时，被证明与一种3'至5'核酸外切酶活性共同纯化。此外，在非变性聚丙烯酰胺凝胶电泳过程中，这两种活性迁移情况相同，并且在甘油梯度离心中共同沉降，表观分子量为105,000。然而，在速度沉降分析后检测到了两种形式的核酸外切酶活性。形式I约占核酸外切酶活性的35%，并与DNA聚合酶相关，而其余活性（形式II）不含DNA聚合酶，分子量约为26,500。形式I核酸外切酶的再沉降产生了与DNA聚合酶相关和不相关的核酸外切酶形式，这表明聚合酶/核酸外切酶发生了解离。核酸外切酶活性（形式I）对N - 乙基马来酰亚胺的抑制有一定抗性，而DNA聚合酶活性则极其敏感。通过十二烷基硫酸钠 - 聚丙烯酰胺活性凝胶电泳后的原位检测表明，形式I和形式II核酸外切酶存在于分子量约为20,000的相似（如果不是相同）多肽中。形式I和形式II核酸外切酶均同样受到氯化钠的抑制，并且需要7.5 mM氯化镁才能达到最佳活性。3'至5'核酸外切酶从3'末端匹配和3'末端错配的引物末端切除脱氧核糖核苷5'-单磷酸。一般来说，根据所有16种可能的匹配和错配末端碱基对组合的Vmax/Km比值确定，核酸外切酶更倾向于水解错配的3'末端核苷酸。这些结果表明，3'至5'核酸外切酶可能参与校对叶绿体DNA聚合酶所产生的错误。
