Molecular hand-off mechanisms during lagging strand replication

滞后链复制过程中的分子传递机制

• 批准号: BB/K021540/1
• 负责人: Panos Soultanas
• 金额: $ 38.67万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK021540%2F1
• 关键词: Molecular; hand; off; mechanisms; during

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.

DNA复制是将一个DNA分子复制成两个相同的DNA分子的过程。它在整个进化过程中的机械水平上是高度保守的。它包括一组高度复杂的生化反应，这些反应由复杂的酶组装体进行，在细胞周期内协调并响应外部和内部细胞信号。在分子水平上对复制过程的了解将提供调节和干预复制的机会;快速分裂的细胞需要在分裂之前复制它们的DNA，并且靶向复制过程的组分可能是治疗癌症和微生物感染的非常强大的策略。靶向致病细菌和病毒的DNA复制是临床现实，但它是药物开发的一个严重不足的领域。DNA复制是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复制的化学过程，我们将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）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。聚合酶不能使用其构建块合成新的DNA，称为脱氧核苷酸三磷酸（简称dNTPs）。相反，亲本DNA首先复制成另一种核酸形式的一小段，称为RNA，RNA是由另一种称为引发酶的酶从其结构单元核糖核苷酸三磷酸（简称NTPs）合成的。然后通过DnaE延伸短RNA片段以形成RNA-DNA杂交片段，然后将其传递给强大且准确的PolC以通过复制亲本模板链进一步延伸。因此，这个过程涉及两种分子传递机制;首先，引发酶合成短RNA并将其传递给DnaE进行初始延伸，其次，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，我们已经纯化了大量的这些蛋白质，并建立了一个新的耦合检测。利用该方法可以同时检测解旋酶（DnaC）解旋亲本双链DNA模板、引物酶（DnaG）合成RNA引物和聚合酶（DnaE）初始延伸RNA引物形成RNA-DNA杂交体的过程。所有三种蛋白质的活动都在这个复合物中协调。我们也有证据表明，PolC纠正DnaE在反式中所犯的错误。使用这种强大的最小耦合分析，我们现在将研究的DnaG-DnaE和DnaE-PolC的手关闭机制的分子细节。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传递机制的结构原理的前所未有的详细了解。

项目成果

SilE is an intrinsically disordered periplasmic "molecular sponge" involved in bacterial silver resistance.

• DOI: 10.1111/mmi.13399
• 发表时间: 2016-09
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Asiani KR;Williams H;Bird L;Jenner M;Searle MS;Hobman JL;Scott DJ;Soultanas P
• 通讯作者: Soultanas P

Engineering a reagentless biosensor for single-stranded DNA to measure real-time helicase activity in Bacillus.

• DOI: 10.1016/j.bios.2014.06.011
• 发表时间: 2014-11-15
• 期刊: BIOSENSORS & BIOELECTRONICS
• 影响因子: 12.6
• 作者: Green, Matthew;Gilhooly, Neville S.;Abedeen, Shahriar;Scott, David J.;Dillingham, Mark S.;Soultanas, Panos
• 通讯作者: Soultanas, Panos

Remodeling and Control of Homologous Recombination by DNA Helicases and Translocases that Target Recombinases and Synapsis.

• DOI: 10.3390/genes7080052
• 发表时间: 2016-08-19
• 期刊: Genes
• 影响因子: 3.5
• 作者: Northall SJ;Ivančić-Baće I;Soultanas P;Bolt EL
• 通讯作者: Bolt EL

Interactions between helicase and primase are crucial for DNA replication in the enteropathogen Clostridium difficile

解旋酶和引物酶之间的相互作用对于肠道病原体艰难梭菌中的 DNA 复制至关重要

• DOI: 10.1101/071829
• 发表时间: 2016
• 作者: Van Eijk E

Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile.

• DOI: 10.1098/rsob.160272
• 发表时间: 2016-12
• 期刊: Open biology
• 影响因子: 5.8
• 作者: van Eijk E;Paschalis V;Green M;Friggen AH;Larson MA;Spriggs K;Briggs GS;Soultanas P;Smits WK
• 通讯作者: Smits WK