Primer Synthesis Kinetics by E. coli Primase

大肠杆菌引物合成动力学

• 批准号: 9600544
• 负责人: Mark Griep
• 金额: $ 24.75万
• 依托单位: University of Nebraska-Lincoln
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9600544&HistoricalAwards=false
• 关键词: Primer; Synthesis; Kinetics; E.; coli

9600544 Griep This is a study of the nucleotide insertion and incorporation kinetics of primase. When errors are incorporated into the DNA during replication, a process during which primase plays a key role, the result is genetic mutation and possibly cancer. Recent studies indicate that primase is the most error-prone enzyme of DNA replication and may very well be the enzyme which misincorporates the most errors into the chromosome. Primase is the single-stranded DNA-dependent RNA polymerase that synthesizes a short (11+1 nucleotide) RNA polymer that serves to initiate DNA synthesis. Primase is required because DNA polymerases are great at elongating DNA but cannot initiate polymers. The primase from E. coli will be studied because it has an especially high specificity during initiation, it prefers to initiate complementary to d(CTG) in the template. This specificity allows one to monitor the length- and sequence-dependence of the primers that are made. Other than this feature though, eukaryotic and bacterial primases exhibit very similar kinetic, structural and binding properties. Primases, like all RNA polymerases, carry out polymer synthesis in three distinct stages, polymer initiation, elongation and termination. Among the nucleic acid polymerases, primases (from bacterial to human) exhibit the highest nucleotide bypass efficiency and the greatest ability to incorporate NTP sugar analogs such as dNTP. Nucleotide bypass is when a polymerase inserts an incorrect nucleotide opposite the template strand and then incorporates that misinsertion by adding the next correct nucleotide. If this were to happen in vivo with anywhere near the efficiency that it occurs in vivo, it might lead to mutation of the genetic material. Current evidence from the proposer's lab suggests that the efficiency of nucleotide bypass changes during the different stages of polymer synthesis. Primase has very high template sequence specificity during initiation but not during elongation or termination. For instance , primase is unlikely to initiate synthesis with a dNTP but will insert a dNMP just as efficiently as a NMP during termination. One possible role for this ability during elongation may be to create a RNA polymer with a 3'-terminal dNMP which may be recognized more effectively by the DNA polymerase. On the other hand, if dNMPs are incorporated more efficiently into the middle of the "RNA primer", then the primer may serve as a better (or worse) target of RNase H or the 5'-3' exonuclease of DNA polymerase I. This study will determine the stage at which primase most efficiently incorporates the correct versus the incorrect dNMPs. The information gained will be used to hypothesize a role for these (mis)incorporated deoxyribonucleotides. %%% The two DNA strands are replicated separately and coordinately. Helicase and DNA polymerase are required for continuous synthesis of the "leading strand" while the "lagging strand" additionally requires primase so that it may be synthesized as numerous short fragments. Each of these fragments begin with an 11+1 nucleotide RNA synthesized by primase that is later excised by other enzymes. Using only primase, its nucleotide substrates, and a DNA template, we have developed a simple assay system to measure template sequence-specific primer synthesis. It was possible to develop this assay system because initiation takes place at a known sequence, d(CTG), and the length of the synthesized primer is 12 nucleotides and greater. In the test tube, pure primase is the most error-prone DNA replication enzyme. We would like to determine when during primer synthesis the most errors occur so that we can hypothesize whether the errors stimulate the excision enzymes and what might be their biological relevance. We will measure the rate of primase errors and determine the effect of distance from the initiation site, of one or several missing sites on the template, of different initiation sequences, and of DNAB helicase on all of these. ***

9600544 griep这是对原始酶的核苷酸插入和掺入动力学的研究。 当复制过程中将误差纳入DNA时，在此过程中，原始酶起关键作用时，结果是基因突变和可能的癌症。最近的研究表明，原始酶是DNA复制的最容易出现错误的酶，很可能是将最大误差误解到染色体中的酶。原始酶是单链DNA依赖性RNA聚合酶，它合成了可启动DNA合成的短（11+1个核苷酸）RNA聚合物。需要原始酶，因为DNA聚合酶在拉长DNA方面很棒，但不能启动聚合物。将研究来自大肠杆菌的早酶，因为它在启动过程中具有特别高的特异性，因此更喜欢在模板中启动与D（CTG）互补的。这种特异性使人们可以监视制作的引物的长度和序列依赖性。除了这一特征外，真核和细菌起点均表现出非常相似的动力学，结构和结合特性。与所有RNA聚合酶一样，原始酶在三个不同的阶段进行聚合物合成，分别是聚合物的启动，伸长和终止。在核酸聚合酶中，根蛋白酶（从细菌到人）表现出最高的核苷酸旁路效率，并且结合了NTP糖类似物（例如DNTP）的最大能力。当聚合酶插入模板链与不正确的核苷酸，然后通过添加下一个正确的核苷酸来纳入误解时，核苷酸旁路是当核苷酸酶插入不正确的核苷酸。 如果这是在体内发生的，并且在体内发生的效率附近，则可能导致遗传物质突变。 提议者实验室的当前证据表明，在聚合物合成的不同阶段，核苷酸绕过的效率变化。启动过程中，原始酶具有很高的模板序列特异性，但在伸长或终止期间没有。 例如，原始酶不太可能与DNTP启动合成，但会在终止期间像NMP一样有效地插入DNMP。 该能力在伸长过程中的可能作用之一可能是创建具有3'-末端DNMP的RNA聚合物，DNA聚合酶可以更有效地识别。 另一方面，如果将DNMP更有效地纳入了“ RNA底漆”的中间，则底漆可能是RNase H或RNase H或5'-3'Exonycleclease的更好（或更糟）的靶标。本研究将确定最有效地合并不正确的DNMPS的Primase的阶段。 获得的信息将用于假设这些（MIS）掺入的脱氧核糖核苷酸的作用。 %% %%这两个DNA链分别复制和协调。在连续合成“前导链”的过程中需要解旋酶和DNA聚合酶，而“滞后链”还需要原始酶，以便可以将其合成为许多短片段。这些片段中的每个片段始于原始酶合成的11+1核苷酸RNA，后来由其他酶切除。仅使用原始酶，其核苷酸底物和DNA模板，我们开发了一个简单的测定系统来测量模板序列特异性引物的合成。 可以开发该测定系统，因为起始是在已知的序列D（CTG）上进行的，而合成底漆的长度为12个核苷酸且更大。 在测试管中，纯培养生酶是最容易出错的DNA复制酶。我们想确定何时在底漆合成过程中出现最多的错误，以便我们可以假设这些误差是否刺激切除酶以及它们的生物学相关性可能是什么。 我们将测量引发液误差的速率，并确定与所有这些对所有这些的距离，一个或几个丢失位点，一个或几个缺失位点的距离，一个或几个缺失位点的效果以及所有这些对DNAB旋转酶的影响。 ***