Mitochondrial DNA polymerase from Drosophila melanogaster embryos: kinetics, processivity, and fidelity of DNA polymerization.
Mitochondrial DNA polymerase from Drosophila melanogaster embryos: kinetics, processivity, and fidelity of DNA polymerization.
复制标题
果蝇胚胎的线粒体 DNA 聚合酶：DNA 聚合的动力学、持续性和保真度。
DOI：
10.1021/bi00416a033
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Kaguni,LS
中科院分区：
文献类型：
--
作者：
Wernette,CM;Conway,MC;Kaguni,LS
生物化学系，密歇根州立大学，东兰辛，密歇根48824接收于1988年2月11日;修订后的Mandarin pt接收于1988年4月11日摘要：从模板-引物利用率、持续合成能力和核苷酸聚合的保真度方面对来自果蝇黑腹胚胎的线粒体DNA聚合酶进行了检测。该酶主要复制单链和双链DNA：DNA合成速率在有缺口的均聚模板poly（dA）·oligo（dT）上最大，而在天然DNA序列的单链DNA模板上观察到最高的底物特异性。动力学实验和DNA合成产物的直接物理分析表明，果蝇DNA聚合酶y通过准进行机制聚合核苷酸。线粒体酶在核苷酸掺入方面表现出高度的准确性，这与果蝇胚胎的复制型DNA聚合酶a几乎相同。因此，近同质的果蝇DNA聚合酶γ的催化性质与在各种动物系统中描述的线粒体DNA合成的体内需要是一致的。果蝇线粒体DNA（mtDNA），1* 像所有的非线粒体mtDNA一样，是环状的线粒体DNA分子（克莱顿，1982）。基因组大小在物种之间从15.7至19.5千碱基对变化，几乎完全是由于称为A+ T区域的单个区域中的序列变异（Wolstenholme等人，1979年）。电子显微镜研究表明，果蝇mtDNA的复制起始于A+ T区，并单向进行（戈达德& Wolsten-holme，1980）。前导DNA链的合成最常见地在互补DNA链合成终止之前完成87-98%，尽管在一小部分分子中，滞后DNA链合成可以更早地开始（Wolstenholme等人，1979;戈达德和Wolstenholme，1980）。因此，果蝇的tDNA是通过一种高度不对称的机制复制的，正如在体内和组织培养条件下在多种生物体中所观察到的那样（克莱顿，1982）。在哺乳动物系统中，复制模式已通过复制中间体的研究详细描述（克莱顿，1982）。虽然通过比较对果蝇或昆虫mtDNA复制知之甚少，但现有数据表明类似的酶要求。为了确定果蝇mtDNA复制的生化和遗传要求，我们描述了从黑腹果蝇胚胎中纯化和部分表征线粒体DNA聚合酶（Poly）（Wernette和Kaguni，1986）。该酶由两个多肽的125 000和35 000道尔顿的SDS-聚丙烯酰胺凝胶电泳判断，是最有可能的异二聚体。而125 000-道尔顿亚基是催化剂的核心，
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 Received February 11, 1988; Revised Manuscript Received April 11, 1988 abstract: The mitochondrial DNA polymerase from embryos of Drosophilamelanogaster has been examined with regard to template-primer utilization, processivity, and fidelity of nucleotide polymerization. The enzyme replicates predominantly single-stranded and double-stranded DNAs: the rate of DNA synthesis is greatest on the gapped homopolymeric template poly (dA)«oligo (dT), while the highest substrate specificity is observed on single-stranded DNA templates of natural DNA sequence. Kinetic experiments and direct physical analysis of DNA synthetic products indicate that the Drosophila DNA polymerase y polymerizes nucleotides by a quasi-processive mechanism. The mitochondrial enzyme demonstrates a high degree of accuracy in nucleotide incorporation which is nearly identical with that of the replicative DNA polymerase a from Drosophila embryos. Thus, the catalytic properties of the near-homogeneous Drosophila DNA polymerase y are consistent with the in vivo requirementsfor mitochondrial DNA synthesis as described in a variety of animal systems.Drosophila mitochondrial DNAs (mtDNAs), 1* like all an-imal mtDNAs, are circular duplexmolecules (Clayton, 1982). The genomesize varies among species from 15.7 to 19.5 ki-lobase pairs almost exclusively as a result of sequence variation in a single region termed the A+ T region (Wolstenholme et al., 1979). Electron microscopic studieshave shown that replication of Drosophila mtDNAs initiates in the A+ T region and proceeds unidirectionally (Goddard & Wolsten-holme, 1980). Synthesis of the leading DNA strand is most frequently 87-98% complete before complementary DNA strand synthesis ensues, although in a small fractionof mol-ecules lagging DNA strand synthesis may be initiated earlier (Wolstenholme et al., 1979; Goddard & Wolstenholme, 1980). Thus, DrosophilamtDNA is replicated by a highly asym-metric mechanism as observed in a variety of organisms, both in vivo and under tissue culture conditions (Clayton, 1982). In mammalian systems, the mode of replication has been described in detail by studies of replication intermediates (Clayton, 1982). Although less is known about Drosophila or insect mtDNA replication by comparison, the available data suggest similar enzymatic requirements. In an effort to define the biochemical and genetic requirements for mtDNA replication in Drosophila, we have described the purification and partial characterization of the mitochondrial DNA polymerase (Pol y) from Drosophila melanogaster embryos (Wernette & Kaguni, 1986). The enzyme consists of two polypeptides of 125 000 and 35 000 daltons as judged by SDS-polyacryl-amide gel electrophoresis, and is most likely a heterodimer. While the 125 000-dalton subunit is the catalytic core of the