Molecular hand-off mechanisms during lagging strand replication

DNA复制是将一个DNA分子复制成两个相同的DNA分子的过程。在整个进化过程中，它在机械水平上是高度保守的。它包括一套高度复杂的生化反应，由复杂的酶组合进行，在细胞周期内协调并响应细胞内外信号。在分子水平上对复制过程的深入了解将为调节和干预复制提供机会；快速分裂的细胞需要在分裂之前复制它们的DNA，而靶向复制过程的组成部分可能是治疗癌症和微生物感染的一种非常有效的策略。靶向致病细菌和病毒的DNA复制是一个临床现实，但它是一个严重未开发的药物开发领域。DNA复制是分子生物学和生物化学广泛应用的基础，为合理设计治疗致病性感染的药物提供了许多潜在靶点。不了解DNA复制的化学过程，我们就无法探索新的药物靶点。一大群致病性和非致病性细菌使用两种不同的酶（DNA聚合酶）复制亲本DNA以形成新生DNA。这两种酶被称为DnaE和PolC。DnaE是一种相对较差的酶，容易出错，而PolC是一种功能强大的酶，保真度极高。聚合酶不能用它的构建块，即脱氧核苷酸三磷酸（简称dNTPs）合成新的DNA。相反，亲本DNA首先以一种核酸的另一种形式被称为RNA的一小段复制，这种核酸是由另一种被称为引物酶的酶从其构建单元核糖核苷酸三磷酸（简称ntp）合成的。短的RNA片段随后被DnaE延长，形成RNA- dna杂交片段，然后传递给强大而精确的PolC，通过复制亲本模板链进一步延长。因此，这一过程涉及两种分子交接机制；首先，引物酶合成短RNA并将其交给DnaE进行初始延伸，然后DnaE形成RNA- dna杂交核酸并将其交给PolC。即使我们对DNA复制有了相对详细的了解，我们仍然对这两种传递机制的分子细节一无所知。在这里，我们旨在研究这些机制并揭示其分子细节。为了做到这一点，我们纯化了大量的这些蛋白质，并建立了一种新的偶联试验。利用该方法，我们可以同时检测亲本双链DNA模板被解旋酶（DnaC）解绕、引物酶（DnaG）合成RNA引物和聚合酶（DnaE）初始延伸形成RNA-DNA杂合体。我们已经确定，这三种蛋白质相互作用，形成一个功能复合物。这三种蛋白质的活动在这个复合体内相互协调。我们也有证据表明PolC纠正了DnaE在翻译中所犯的错误。利用这种强大的最小耦合分析，我们现在将研究DnaG-DnaE和DnaE-PolC交接机制的分子细节。我们还将建立相互作用蛋白质的结构模型，以获得对支撑这两种交接机制的结构原理的前所未有的详细了解。

DNA replication is the process of copying one DNA molecule to form two identical ones. It is highly conserved at the mechanistic level across evolution. It comprises a highly complex set of biochemical reactions carried out by intricate enzyme assemblies, coordinated within the cell cycle and in response to external and internal cellular signals. Insights into the replication processes at the molecular level will provide opportunities to modulate and intervene in replication; rapidly dividing cells need to replicate their DNA prior to dividing, and targeting components of the replication process is potentially a very powerful strategy in the treatment of cancer and microbial infections. Targeting DNA replication of pathogenic bacteria and viruses is a clinical reality but it is a grossly underexplored area of drug development. DNA replication is fundamental to a huge range of molecular biological and biochemical applications, and provides many potential targets for rational drug design in the treatment pathogenic infections. Without understanding the chemistry of DNA replication we will not be able to explore new drug targets.A large group of pathogenic and non-pathogenic bacteria use two different enzymes (DNA polymerases) to copy the parental DNA to form nascent DNA. These two enzymes are known as DnaE and PolC. DnaE is a relatively poor enzyme prone to making mistakes, while PolC is a powerful enzyme with extremely high fidelity. The polymerases cannot synthesize new DNA using its building blocks, known as deoxynucleotide tri-phosphates (dNTPs for short). Instead, the parental DNA is first copied in a short stretch of an alternative form of a nucleic acid, known as RNA, which is synthesized from its building blocks ribonucleotide tri-phosphates (NTPs for short) by another enzyme known as primase. The short RNA stretch (fragment) is then extended by DnaE to form an RNA-DNA hybrid fragment which is then handed off to the powerful and accurate PolC to be extended further by copying the parental template strand. This process, therefore, involves two molecular hand-off mechanisms; First the primase synthesizes the short RNA and hands it off to DnaE for initial extension and second the DnaE forms the RNA-DNA hybrid nucleic acid and hands it off to PolC. Even with all our relatively detailed knowledge of DNA replication we still know nothing about the molecular details of these two hand-off mechanisms. Here, we aim to study these mechanisms and reveal their molecular details. In order to do this we have purified large quantities of these proteins and set up a novel coupled assay. With this assay we can detect simultaneously, unwinding of the parental double stranded DNA template by the enzyme helicase (DnaC), synthesis of the RNA primer by the enzyme primase (DnaG) and initial extension of the RNA primer to form the RNA-DNA hybrid by the enzyme polymerase (DnaE).We have established that these three proteins interact with each other to form a functional complex. The activities of all three proteins are coordinated within this complex. We also have evidence suggesting that PolC corrects the mistakes made by DnaE in trans. Using this powerful minimal coupled assay we will now study the molecular details of the DnaG-DnaE and DnaE-PolC hand off mechanisms. We will also build structural models of the interacting proteins to gain unprecedented detailed understanding of the structural principles that underpin these two hand-off mechanisms.