Characterization of the 5' to 3' exonuclease associated with Thermus aquaticus DNA polymerase.
Characterization of the 5' to 3' exonuclease associated with Thermus aquaticus DNA polymerase.
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与栖热菌 DNA 聚合酶相关的 5 至 3 核酸外切酶的表征。
DOI：
10.1093/nar/18.24.7317
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发表时间：
1990
影响因子：
14.9
通讯作者：
Mosbaugh,DW
中科院分区：
文献类型：
--
作者：
Longley,MJ;Bennett,SE;Mosbaugh,DW
研究表明，水藻dna聚合酶含有相关的5′至3′外切酶活性。在蔗糖梯度离心过程中，聚合酶和外切酶活性共沉淀，分子量为72,000。使用一种新颖的情境活性凝胶程序同时检测这两种活性，我们在非变性或变性聚丙烯酰胺凝胶电泳中观察到DNA聚合酶和外切酶在一个条带上：因此，DNA聚合酶和外切酶活性存在于同一多肽中。经sds -聚丙烯酰胺凝胶电泳测定，该酶表观分子量为92000。该核酸外切酶需要二价阳离子（mgcl2或MnCl2）， pH最适值为9.0，主要从双链DNA中切除脱氧核糖核苷5 ' -单磷酸。热变性DNA和游离寡核苷酸（24-mer）都不是核酸外切酶活性的有效底物。与含有5 ' -羟基化残基的相同底物相比，退火到M13mp2 DNA的5 ' -磷酸化寡核苷酸（24-mer）的水解速度大约快两倍。与双链DNA相同的5 ‘端相比，缺口的5 ’端残基的水解是三倍。5 ‘至3 ’外切酶活性似乎与DNA聚合酶协同作用，以促进nick翻译DNA合成反应。
Thermus aquaticusDNA polymerase was shown to contain an associated 5′ to 3′ exonuclease activity. Both polymerase and exonuclease activities cosedimented with a molecular weight of 72,000 during sucrose gradient centrifugation. Using a novelin situactivity gel procedure to simultaneously detect these two activities, we observed both DNA polymerase and exonuclease in a single band following either nondenaturing or denaturing polyacrylamide gel electrophoresis: therefore, DNA polymerase and exonuclease activities reside in the same polypeptide. As determined by SDS-polyacrylamide gel electrophoresis this enzyme has an apparent molecular weight of 92,000. The exonuclease requires a divalent cation (MgCl2or MnCl2), has a pH optimum of 9.0 and excises primarily deoxyribonucleoside 5′-monophosphate from double-stranded DNA. Neither heat denatured DNA nor the free oligonucleotide (24-mer) were efficient substrates for exonuclease activity. The rate of hydrolysis of a 5′-phosphorylated oligonucleotide (24-mer) annealed to M13mp2 DNA was about twofold faster than the same substrate containing a 5′-hydroxylated residue. Hydrolysis of a 5′-terminal residue from a nick was preferred threefold over the same 5′-end of duplex DNA. The 5′ to 3′ exonuclease activity appeared to function coordinately with the DNA polymerase to facilitate a nick translational DNA synthesis reaction.