Real-time Monitoring of Nucleotide Excision of Molecular Beacon Catalyzed by Klenow Fragment

Klenow fragment（KF） uses the activity of a separate exonuclease to excise nucleotide, which is a crucial step in DNA replication and repair. Here is a novel sensitive and convenient method introduced for real-time monitoring nucleotide excision by KF with a molecular beacon as a detecting probe in a homogeneous solution. This method, which overcomes the drawbacks of traditional methods such as discontinuity, time consuming and low sensitivity, was used to assay KF activity and the detection limit reached up to 0.4 U/mL. In addition, the method was applied to investigating the effects of metal ions and chemical drugs on the reaction. The results demonstrate that it is a potential high-throughput assay for screening inhibitors and activity analysis of KF in vitro.

New insight into the mechanism of DNA polymerase I revealed by single-molecule FRET studies of Klenow fragment

We use single-molecule FRET and newly-developed D-loop techniques to investigate strand displacement activity of Klenow fragment(exo-)of DNA polymerase I in DNA sequences rich in guanine and cytosine(GC)bases.We find that there exist in the FRET traces numerous ascending jumps,which are induced by the backsliding of Klenow fragment on DNA chains.Our measurements show that the probability of backsliding is closely related to the GC-richness and d NTP concentration:increasing the GC-richness leads to an increase in the backsliding probability,and increasing the d NTP concentration however leads to a decrease in the backsliding probability.These results provide a new insight into the mechanism of DNA polymerase I.

Acceleration of DNA Replication of Klenow Fragment by Small Resisting Force

DNA polymerases are an essential class of enzymes or molecular motors that catalyze processive DNA syntheses during DNA replications. A critical issue for DNA polymerases is their molecular mechanism of processive DNA replication. We have proposed a model for chemomechanical coupling of DNA polymerases before, based on which the predicted results have been provided about the dependence of DNA replication velocity upon the external force on Klenow fragment of DNA polymerase I. Here, we performed single molecule measurements of the replication velocity of Klenow fragment under the external force by using magnetic tweezers. The single molecule data verified quantitatively the previous theoretical predictions, which is critical to the chemomechanical coupling mechanism of DNA polymerases. A prominent characteristic for the Klenow fragment is that the replication velocity is independent of the assisting force whereas the velocity increases largely with the increase of the resisting force,attains the maximum velocity at about 3.8 pN and then decreases with the further increase of the resisting force.

DNA聚合酶-ⅠKlenow片段原位检测缺血性神经元的DNA早期断裂

目的 探讨脑缺血 30min后DNA断裂损伤的发生时间。方法 用DNA聚合酶 ⅠKlenow大片段原位检测缺血性神经元的DNA早期断裂。结果 缺血再灌注 1h顶叶皮层、梨状皮层出现DNA断裂 ,再灌注 2h尾壳核出现DNA断裂 ,再灌注 2 4h ,各个区域的DNA断裂均达到顶峰。结论 DNA断裂损伤可能是缺血神经元改变的早期事件之一。

重组Klenow-HSA融合蛋白的构建、纯化及其稳定性

目的 :提高Klenow酶的热稳定性和储藏稳定性。方法 :利用PCR的方法扩增DH5α染色体DNA ,构建编码Klenow和人血清白蛋白第三功能区 (HSA D3)的融合蛋白表达质粒 ,并在大肠杆菌中获得高效表达。分别对Klenow酶和Klenow HSA融合蛋白进行纯化 ,并进行活性及稳定性的研究。结果 :发现Klenow HSA融合蛋白在大肠杆菌中呈可溶性表达。体外实验证实这种融合蛋白具有与Klenow聚合酶相同的活性 ,其稳定性明显提高。结论 :融合蛋白确实能增加Klenow酶的热稳定性和储存稳定性 。

大鼠局灶性脑缺血再灌注早期DNA单链断裂损伤及神经元形态变化

目的 探讨大鼠大脑中动脉阻塞再灌注 (MCAO/ R)早期 DNA断裂损伤的情况及其意义。方法 使用尼氏染色方法观察再灌注早期损伤区域神经元数量和形态的变化 ,使用 DNA聚合酶 - Klenow片段原位检测 DNA的断裂损伤。结果 再灌注 6 h损伤区域部分神经元出现形态变化时 ,可见少量 Klenow阳性细胞 ;再灌注2 4 h损伤区域大量神经元出现形态变化时 ,Klenow阳性细胞数量陡然显著增加 (P<0 .0 1) ;再灌注 72 h损伤区域神经元数量明显减少时 ,Klenow阳性细胞数量较 2 4 h时有所下降 (P<0 .0 1)。结论 (1)局灶性脑缺血再灌注 2 4 h时 ,数量达到峰值的 Klenow阳性细胞中多数应为单链 DNA断裂的细胞 ,并推测这一损伤形式可能是由自由基引发的一系列 DNA损伤过程中的一个重要环节 ;(2 )单链 DNA断裂大量产生的同时 ,损伤区域大量神经元出现病理形态变化 ,说明神经元的不可逆损伤过程在单链 DNA断裂时可能就已经发生。

用部分补平法克隆乙型肝炎病毒preS/S基因

目的探讨利用Ｋｌｅｎｏｗ大片段对ＤＮＡ末端作部分补平而使非匹配粘端形成匹配粘端的可行性。方法用 ＢａｍＨＩ、ＸｂａｌＩ消解载体 ｐｃＤＮＡ３，用 ＢｇｌＨ、Ｈｉｎｄ Ⅲ从含乙型肝炎病毒全基因组质粒中切取 ＨＢＶｐｒｅＳ／Ｓ基因片段。 ｐｃＤＮＡ３的 ＸｂａｌＩ及 ｐｒｅＳ／Ｓ基因的 Ｈｉｎｄ Ⅲ切口端经 Ｋｌｅｎｏｗ大片段作部分补平后形成二碱基的互补粘端。连接修饰 后的ｐｒｅＳ／Ｓ基因片段与ｐｃＤＮＡ３并转化后筛选阳性克隆。结果限制性内切酶分析显示ｐｒｅＳ／Ｓ基因被定向克隆于载 体ｐｃＤＮＡ３的预定位点。结论部分补平法是连接非匹配ＤＮＡ粘性末端的较好选择。

DNA聚合酶Ⅰ的功能与结构的发现

本文具体介绍了DNA聚合酶Ⅰ的功能与结构的发现过程,包括聚合酶活性中心模型的提出;聚合酶分子量的测定;聚合酶的分离及其活性片段的获得;聚合酶Klenow片段晶体结构的测定以及DNA结合部位与活性位点的确立;Klenow片段结合DNA的晶体结构以及噬菌体T7DNA复制的晶体结构的测定,在测定这些复合物共晶体的基础上,提出了DNA与聚合酶结合的右手模型。

利用套叠PCR技术合成人胰岛素原基因的临床研究

目的设计并分子克隆适宜于大肠杆菌表达系统的人胰岛素原基因。方法以美国国家基因库(GenBank)发布的人胰岛素原的氨基酸序列为模板,参考大肠杆菌表达系统的密码子偏好性以及所用表达载体(pGEX-3x)的特点,经计算机分析、设计人胰岛素原基因为8个寡核苷酸片段,以重叠延伸PCR(SOE-PCR)技术体外延伸获得完整的人胰岛素原基因,分子克隆后测定所得基因的核苷酸序列。结果经限制性内切酶分析确定为重组克隆的质粒进行核苷酸序列分析,结果表明,我们所克隆的基因序列完全符合我们先前的设计。结论SOE-PCR技术可用于体外寡核苷酸片段的延伸并获得完整长度的基因。TaqDNA聚合酶延伸反应前以Klenow片段处理可提高较短覆盖末端或较低专一性末端正确延伸的成功率。

Single-strand DNA damages and its relationship with morphological change of neurons at early stage of rat cerebral ischemia/reperf usion

Objective：To discuss the DNA-strand breaks at early stage of middle cerebral artery occlusion/reperfusion (MCAO/R). Methods: Neurons number and morphologic change were observed by Nissl stain method, and DNA strand breaks were in situ detected by using DNA polymerase- I Klenow fragment-mediat-ed nick end-labelling method (Klenow method). Results: Six hours after reperfusion, a few neurons in dam-aged regions appeared morphologic changes while a few Klenow-positive cells were detected (P<0. 01). Twenty-four hours after reperfusion lots of neurons showed morphologic change while the number of Klenow-positive cells immediately and remarkably increased (P<0. 01). Seventy-two hours after reperfusion the number of neurons decreased significantly and the number of Klenow-positive cells was also less than that in 24 h (P<0. 05). Conclusion: ① 24 h after reperfusion when the number of Klenow-positive cells reached peak value, DNA single-strand breaks (SSBs) took place in many Klenow-positive cells, and presumed that DNA SSBs might be an important step in DNA-damage procession which might be induced by free radicals. ② At the same time when lots of DNA SSBs were produced, many neurons in the damaged regions showed morphological change, which indicated that lots of neurons had already progressed to irreversible damages when DNA SSBs took place.

基于金纳米颗粒信号放大效应电化学检测DNA聚合酶

基于金纳米颗粒(AuNPs)比表面积大、尺寸小和能够承载大量DNA片段的特点,建立了一种免标记、简便、快速检测DNA聚合酶Klenow fragment exo-(KF-)的电化学方法.首先将巯基化的DNA引物片段修饰在金电极上,然后加入模板DNA链以及修饰有报告DNA链的金纳米颗粒(AuNPs-DNA),模板DNA链能同时与DNA引物片段和修饰在AuNPs上的报告DNA链进行互补杂交形成"三明治"结构,从而将AuNPs-DNA修饰在电极表面;当加入电活性物质钌铵(RuHex)后,RuHex可通过静电吸附作用结合在DNA上.AuNPs上修饰的报告DNA链能够吸附大量RuHex,导致电化学信号放大.当加入脱氧核糖核苷三磷酸(dNTPs)以及KF-聚合酶后,引物片段发生延伸反应,将与模板DNA链杂交的AuNPs-DNA竞争下来,带走大量的RuHex,使电信号降低,从而实现对聚合酶的检测.实验结果表明,利用该方法可以检测到5 U/mL的KF-.

噬菌体RB69 DNA聚合酶的表达、分离纯化和其聚合反应的稳态动力学研究

噬菌体ＲＢ６９外切酶活性缺失的ＤＮＡ 聚合酶突变体（Ｄ２２２Ａ／Ｄ３２７Ａ）在大肠杆菌细胞中表达，表达量达细胞蛋白总量的６９％ ．表达后经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＦａｓｔＦｌｏｗ ， Ｓｏｕｒｃｅ ３０Ｑ 和ＨＴＰ三步分离纯化，纯度可达９９％ 以上．随后测定了该酶利用５种ｄＮＴＰ为底物进行聚合反应的酶促动力学常数（Ｋｍ 和Ｋｃａｔ），结果表明该酶利用ｄＵＴＰ的能力与利用ｄＴＴＰ的能力相近，Ｋｍ （ｄＴＴＰ）和Ｋｍ（ｄＵＴＰ）均较高于其它３种脱氧核苷酸的Ｋｍ （ｄＡＴＰ， ｄＣＴＰ， ｄＧＴＰ），推测其Ｋｍ 值的差异主要来源于Ｔ／Ｕ 碱基本身，而并非全部由ＧＣ碱基配对与ＡＴ碱基配对之间的氢键作用力的强弱差别所决定．