Properties of two forms of DNA polymerase delta from calf thymus.
Properties of two forms of DNA polymerase delta from calf thymus.
复制标题
小牛胸腺中两种形式的 DNA 聚合酶 δ 的特性。
DOI：
10.1021/bi00372a006
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Bambara,RA
中科院分区：
文献类型：
--
作者：
Wahl,AF;Crute,JJ;Sabatino,RD;Bodner,JB;Marraccino,RL;Harwell,LW;Lord,EM;Bambara,RA
1986年8月1日收到的修订版摘要;纯化的小牛胸腺DNA聚合酶I和II各自具有相关的3 '至5'外切核酸酶，但在尺寸、生化动力学参数和DNA引发酶的存在方面类似于DNA聚合酶a [Crute，JJ，Wahl，A. F.、& Bambara，R. A.（1986）Biochemistry 25，26-36]，在此我们证明了聚合酶和核酸外切酶与每种形式的功能关联。此外，我们表明，核酸外切酶可以从DNA聚合酶I解离，但似乎不能从DNA聚合酶II中去除。聚合酶I、II和a对抑制剂aphidicolin同样敏感，表明活性位点结构相似。与DNA聚合酶a和II相比，DNA聚合酶I对2-（pn-丁基苯胺基）-2 '-脱氧腺苷S'-三磷酸（BuAdATP）或N2-（pn-丁基苯基）-2 '-脱氧鸟苷S'-三磷酸（BuPdGTP）具有中等敏感性。II酶的DNA引发酶活性对BuAdATP不敏感，而1.0 µ该抑制剂将使a和I酶的DNA引发酶活性降低约50%。两种有效抑制DNA聚合酶a的单克隆抗体对DNA聚合酶I只有轻微的抑制作用，而对DNA聚合酶II则无效。DNA聚合酶II先前已被发现是几乎无活性的核酸酶处理的小牛胸腺DNA，相对于其活性的均聚DNA。我们发现，添加纯化的小牛组蛋白或亚精胺可以大大增强这种酶在活化的小牛DNA上的合成。最近使用离子交换和底物亲和色谱法从小牛胸腺中纯化了两种形式的DNA聚合酶（I和II）（Crute等，1986年a）。它们是含有3 '至5'核酸外切酶活性的聚合酶，并且通过色谱行为和模板偏好而彼此不同。基于它们与先前描述的聚合酶的相似性，它们被指定为聚合酶类（Byrnes等人，1976; Lee等人，1984; Byrnes，1984）。对聚合酶的检查表明，它们与小牛DNA聚合酶a具有许多共同性质，我们使用免疫亲和方法纯化了小牛DNA聚合酶a（Wahl等人，1984年）。这三种酶，DNA聚合酶a、I和II，具有相似的分子量和轴比，具有相关的DNA引发酶活性，显示出相似的进行性合成程度，并被ATP刺激。该研究得到了美国国立卫生研究院赠款GM 24441（RAB）和CA 28322（EML）以及癌症中心核心赠款5-P30-CA 11198-16的支持。
Revised Manuscript Received August 1, 1986 abstract; Purified calf thymus DNA polymerases I and II each have an associated 3'to 5'exonuclease but otherwise resemble DNA polymerase a in size, biochemical kinetic parameters, and the presence of DNA primase [Crute, JJ, Wahl, A. F., & Bambara, R. A.(1986) Biochemistry 25, 26-36], Here we demonstrate a functional association of polymerase and exonuclease with each form. Furthermore, we show that the exonuclease can be dissociated from DNA polymerase I but does not appear to be removable from DNA polymerase II. Polymerases I, II, and a are equally sensitiveto the inhibitor aphidicolin, suggesting a similarity in active site structure. In comparison with DNA polymerase a and II, DNA polymerase I has intermediate sensitivity to 2-(pn-butylanilino)-2'-deoxyadenosine S'-triphosphate (BuAdATP) or N2-(pn-butylphenyl)-2'-deoxyguanosine S'-triphosphate (BuPdGTP). The activity of the DNA primase of the II enzyme is insensitive to BuAdATP whereas 1.0 µ of this inhibitor will decrease the activity of the DNA primase of the a and I enzymes approximately 50%. Two monoclonal antibodies that potently inhibit DNA polymerase a are only slightly inhibitory to DNA polymerase I and are ineffective at inhibitingDNA polymerase II. DNA polymerase II had been previously found to be nearly inactive on nuclease-treated calf thymus DNA, relative to its activity on homopolymeric DNA. We find that addition of purified calf histone proteins or spermidine can greatly enhance synthesis by this enzyme on activated calf DNA. have recently purified two forms of DNA polymerase (I and II) from calf thymususing ion-exchange and substrate affinity chromatography (Crute et al., 1986a). They are polymerases that contain a 3'to 5'exonuclease activity and differ from each other by chromatographic behavior and template preference. They have been designated-class polymerases on the basis of their similarities of the polymerase described earlier (Byrnes et al., 1976; Lee et al., 1984; Byrnes, 1984). Examination of the polymerases has revealed that they share many properties with calf DNA polymerase a, which we have purified using an immunoaffinity method (Wahl et al., 1984). The three enzymes, DNA polymerases a, I, and II, have similar molecular weight and axial ratio, have an associated DNA primase activity, display similar extents of processive synthesis, and are stimulated by ATP f The research was supported by National Institutes of Health Grants GM 24441 to RAB and CA28322 to EML and Cancer Center Core Grant 5-P30-CA 11198-16.