大肠杆菌DNA聚合酶Ⅰ的“碱基选择”作用  

Base Selection by E. colt DNA Polymerase I

作　　者：陆长德[1] El.,WS 

机构地区：[1]中国科学院上海生物化学研究所

出　　处：《生物化学与生物物理学报》1995年第6期593-601,共9页

基　　金：美国NIH资助

摘　　要：用双脱氧核苷酸研究了大肠杆菌ＤＮＡ聚合酶Ⅰ的Ｋｌｅｎｏｗ片段的“碱基选择”作用．当引物末端为３’ＯＨ时，Ｍｎ２＋代替Ｍｇ２＋对ｄＮＴＰ的Ｋｍ影响不大，而使ｄｄＮＴＰ的Ｋｉ大大降低，非配对的ｄｄＮＴＰ仍无抑制作用。当引物末端为ｄｄＮＭＰ且与模板配对时是ＤＮＡ合成的抑制剂，与模板配对的下一个ｄＮＴＰ可增强这一抑制作用，Ｍｎ２＋代替Ｍｇ２＋使ｄＮＴＰ的诱导作用大大增强而ｄｄＮＴＰ无诱导作用。这些结果表明引物末端对于酶选择核苷酸起着重要作用，ＤＮＡ聚合酶Ⅰ选择核苷酸的过程应是个有序的构象变化的过程。下一互补核苷酸的结合抑制３＇→５＇外切活性对引物末端ｄｄＮＭＰ的水解作用为此提供了又一证据。Dideoxynucleotides have been used to study the base selection by E. coli DNA polymerase I. With a deoxy-terminated Primer/template, the substitution of Mn2+for Mg2+ results in a marked decrease of the K1 of ddNTP, whereas the Km of dNTP remains unchanged. Noncomplementary ddNTPs still show no inhibition. Dideoxyterminated Primer/templates are found to competitively inhibit DNA synthesis only when the ddNMP terminus is complementary to the inhibitol DNA template. Furthermore, a dNTP that is complementary to the competitor DNA template potentiates this inhibition and the substitution of Mn2+ for Mg2+ results in a marked increase in the inducing ability of dNTP but not ddNTP. These results suggest that the primer torminus plays a crucial role in base selection. It is proposed that the high specificity of base selection by DNA polymerase I is achieved by sequential conformational changes. The inhibition of the hydrolysis of 3'ddNMP of the primer by 3'→5' exonuclease upon the binding of the next incoming dNTP Provides another evidence for the secondary conformational changes of the enzyme.

关 键 词：DNA聚合酶I 碱基选择 大肠杆菌 

分 类 号：Q555.02[生物学—生物化学]