Longley M J, Mosbaugh D W
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331.
Biochemistry. 1991 Mar 12;30(10):2655-64. doi: 10.1021/bi00224a014.
We have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined 32P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their [32P]DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase beta were found to be similar under both in vitro and in situ conditions. With 3'-terminally matched and mismatched [32P]DNA substrates in the same activity gel, DNA polymerase and/or 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. In addition, use of matched and mismatched DNA primers permitted the uncoupling of mismatch excision and chain extension steps. Activities first detected in nondenaturing activity gels as either multifunctional or multimeric enzymes were also identified in denaturing activity gels, and assignment of activities to specific polypeptides suggested subunit composition. Furthermore, DNA substrates cast within polyacrylamide gels were successfully modified by the exogenous enzymes polynucleotide kinase and alkaline phosphatase before and after in situ detection of E. coli DNA ligase activity, respectively. Several restriction endonucleases and the tripeptide (Lys-Trp-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.
我们使用一种新型的聚丙烯酰胺活性凝胶电泳方法检测了DNA糖基化酶、核酸内切酶、核酸外切酶、DNA聚合酶和DNA连接酶的原位活性。通过含有与M13 DNA退火的特定32P标记寡核苷酸的天然或SDS-聚丙烯酰胺凝胶分离DNA代谢酶。电泳后，这些酶催化原位反应，其[32P]DNA产物通过变性DNA测序凝胶的二维电泳从凝胶中分离出来。通过检测修饰的（降解或延长的）寡核苷酸链来定位各种酶的活性。发现诺维科夫肝癌DNA聚合酶β在体外和原位条件下的催化和物理性质相似。在同一活性凝胶中使用3'-末端匹配和不匹配的[32P]DNA底物，检测并表征了大肠杆菌DNA聚合酶I（大片段）、DNA聚合酶III（全酶）和核酸外切酶III的DNA聚合酶和/或3'至5'核酸外切酶活性。此外，使用匹配和不匹配的DNA引物可以使错配切除和链延伸步骤解偶联。在非变性活性凝胶中最初检测为多功能或多聚体酶的活性在变性活性凝胶中也得到了鉴定，将活性分配给特定多肽表明了亚基组成。此外，在原位检测大肠杆菌DNA连接酶活性之前和之后，分别用外源酶多核苷酸激酶和碱性磷酸酶成功修饰了聚丙烯酰胺凝胶中浇铸的DNA底物。几种限制性核酸内切酶和作为脱嘌呤/脱嘧啶核酸内切酶的三肽（Lys-Trp-Lys）能够扩散到凝胶中并修饰DNA。在活性凝胶中创建中间底物的这种能力在描绘DNA复制和修复途径的步骤方面可能被证明极其有用。
