Molecular hand-off mechanisms during lagging strand replication

DNA复制是复制一个DNA分子形成两个完全相同的分子的过程。在整个进化过程中，它在机械论层面上是高度保守的。它包括一系列高度复杂的生化反应，由错综复杂的酶组合进行，在细胞周期内协调，并对外部和内部细胞信号做出反应。在分子水平上对复制过程的洞察将提供调节和干预复制的机会；快速分裂的细胞在分裂前需要复制它们的DNA，而针对复制过程的组件在治疗癌症和微生物感染方面可能是一个非常有效的策略。以致病细菌和病毒的DNA复制为靶点是临床现实，但这是药物开发中一个严重未被探索的领域。DNA复制是广泛的分子生物学和生物化学应用的基础，并为治疗病原性感染的合理药物设计提供了许多潜在的靶点。如果不了解DNA复制的化学原理，我们将无法探索新的药物靶点。一大群致病和非致病细菌使用两种不同的酶(DNA聚合酶)来复制亲本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，具有极高的保真度。聚合酶不能使用它的组成部分，即所谓的脱氧核苷酸三磷酸(DNTPs)合成新的DNA。取而代之的是，父母的DNA首先在一种被称为RNA的替代形式的一小段核酸中复制，RNA是由其构建块三磷酸核糖(NTPs)由另一种被称为Primase的酶合成的。然后，短RNA延伸(片段)被DNAE延伸以形成RNA-DNA杂交片段，该片段随后被移交给强大且准确的Polc，以通过复制亲本模板链来进一步延伸。因此，这一过程涉及两种分子传递机制：第一种是启动酶合成短RNA并将其传递给DNAE进行初始延伸；第二种是DNAE形成RNA-DNA杂交核酸并将其传递给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，我们的目标是研究这些机制，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分析方法。利用该方法可以同时检测到解旋酶(DNAC)解开亲本双链DNA模板、酶引物酶(DNAG)合成RNA引物和酶聚合酶(DNAE)起始延伸RNA-DNA杂交体，这三种蛋白质相互作用形成一个功能复合体。在这个复合体中，所有三种蛋白质的活性都是协调的。我们也有证据表明Polc在TRANS中纠正了DNAE所犯的错误。利用这一强大的最小偶联分析，我们现在将研究DNAG-DNAE和DNAE-Polc移交机制的分子细节。我们还将建立相互作用蛋白质的结构模型，以前所未有的详细了解支撑这两种移交机制的结构原理。

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.