Primer Synthesis Kinetics by E. coli Primase

这是对引物酶核苷酸插入和结合动力学的研究。当错误在复制过程中被纳入DNA时（在这个过程中引物酶起着关键作用），结果是基因突变和可能的癌症。近年来的研究表明，引物酶是DNA复制过程中最容易出错的酶，也很可能是染色体错误整合最多的酶。引物酶是单链DNA依赖的RNA聚合酶，可合成短（11+1核苷酸）RNA聚合物，用于启动DNA合成。引物酶是必需的，因为DNA聚合酶能很好地延长DNA，但不能引发聚合物。大肠杆菌引物酶在启动过程中具有特别高的特异性，它倾向于启动模板中的互补d（CTG）。这种特异性使人们能够监测所制引物的长度和序列依赖性。除了这个特征，真核生物和细菌的引物表现出非常相似的动力学、结构和结合特性。像所有RNA聚合酶一样，引物酶在三个不同的阶段进行聚合物合成，即聚合物起始、延伸和终止。在核酸聚合酶中，引物酶（从细菌到人类）表现出最高的核苷酸旁路效率和结合NTP糖类似物（如dNTP）的最大能力。核苷酸绕过是指聚合酶在模板链的对面插入一个错误的核苷酸，然后通过添加下一个正确的核苷酸来整合这个错误的插入。如果这种情况在体内以接近体内的效率发生，它可能会导致遗传物质的突变。目前来自提议者实验室的证据表明，核苷酸旁路的效率在聚合物合成的不同阶段发生变化。引物酶在起始阶段具有非常高的模板序列特异性，而在延伸和终止阶段则没有。例如，引物酶不太可能用dNTP启动合成，但在终止过程中插入dNMP和NMP一样有效。这种能力在延伸过程中的一个可能的作用可能是产生具有3'端dNMP的RNA聚合物，这可能被DNA聚合酶更有效地识别。另一方面，如果dnmp更有效地结合到“RNA引物”的中间，那么该引物可能作为RNase H或DNA聚合酶i的5‘-3’外切酶的更好（或更差）靶标。本研究将确定引物酶在哪个阶段最有效地结合正确的dnmp和不正确的dnmp。获得的信息将用于假设这些（错误）结合的脱氧核糖核苷酸的作用。这两条DNA链分别协调地复制。“前导链”的连续合成需要解旋酶和DNA聚合酶，而“后导链”还需要引物酶才能合成成许多短片段。每个片段都以引物酶合成的11+1核苷酸RNA开始，随后被其他酶切除。仅使用引物酶，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合成。由于起始发生在一个已知的序列d（CTG）上，并且合成的引物长度为12个核苷酸或更大，因此开发该检测系统是可能的。在试管中，纯引物酶是最容易出错的DNA复制酶。我们想要确定在引物合成过程中什么时候错误最多，这样我们就可以假设这些错误是否刺激了切除酶，以及它们的生物学相关性。我们将测量引物酶错误率，并确定与起始位点的距离，模板上的一个或几个缺失位点，不同的起始序列以及DNAB解旋酶对所有这些的影响。＊＊＊

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. ***