Molecular hand-off mechanisms during lagging strand replication

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.

Processing of the DNA during replication and repair proceeds through intricately choreographed entry and exit of individual proteins which has been termed "trading places" on the DNA or "molecular hand-off". A critical implication of molecular hand-off is that the protein complexes required at each step are successively remodelled to enable the next step in the process to be performed. This key characteristic provides the flexibility to repair a wide variety of lesions in the DNA template, multiple levels of regulation, and the recycling of commonly used proteins. Another hallmark of these functions is the major role of modular proteins (polypeptides composed of multiple domains that are connected by flexible linkers). These modular proteins physically interact with each other through contacts in one or more of these domains or, in some cases, in linkers between domains. Critical for our understanding of the replisome is to reveal how these modular activities are coordinated during DNA synthesis and repair.

The replication system of the Gram +ve model bacterium Bacillus subtilis (and the rest of the Firmicutes) is more similar than Escherichia coli to the eukaryotic system. Like the eukaryotes, B. subtilis also uses two essential polymerases DnaE and PolC. PolC is the main processive, high fidelity polymerase on the leading and lagging strands but cannot extend the RNA primers. Initial extension of the RNA primers is carried out by the relatively poorly processive and error-prone lagging strand specific DnaE which then hands-off the RNA-DNA fragments to PolC for processive and accurate extension. There is no understanding of how DnaG hands off the RNA primers to DnaE and how DnaE subsequently hands off the RNA-DNA hybrid to PolC.

I have developed a minimal coupled helicase-primase-polymerase assay using purified recombinant proteins which I will use to study the molecular details of the primase-polymerase and polymerase-polymerase hand-off mechanisms.

DNA replication is the process of copying a double stranded DNA molecule to synthesize two identical new copies. It is the process that maintains and drives all life on our planet and as such it is highly conserved at the mechanistic level across evolution. The task of replicating astronomical numbers of nucleotides safely and accurately is considerable. A large number of proteins have evolved to deal with different challenges faced during the replication process. Understanding the basic chemistry of DNA replication has led to the development of the ground-breaking techniques such as the "Sanger termination dideoxy sequencing" that has led to the explosion of molecular biology and revolutionized our understanding of genomes and our ability to manipulate genomes. Such work paved the way to the next generation of polysequencing culminating in the pivotal achievement of sequencing the entire human genome. Cloning of genes and polymerase chain reaction (PCR) technology used in almost every aspect of modern life sciences research require DNA replication. In fact PCR is simply region-specific replication on a grand scale. Therefore, understanding fundamental biological processes, such as DNA replication, through basic biological research at the molecular and chemical levels is essential to developing universally applicable technologies and using them effectively across all life sciences. The BBSRC fully recognizes the importance of basic research and has set in its recent strategic review one of its core strategic aims to "advance fundamental understanding of complex biological processes". Our recent research on collisions between replication and transcription that was published in the journal Nature was featured in the BBSRC Business Spring 2011 edition and in that feature Prof. Douglas Kell, the BBSRC Chief Executive, commented, "The interplay between gene expression, DNA replication and the prevention of DNA damage is an example of a tenet of biology that has the potential to touch on research right across BBSRC's portfolio and beyond". Beyond the development of DNA replication based technologies the crucial importance of DNA replication in human health is undisputed. When DNA replication goes wrong, by incorporating mutations and/or by losing regulatory controls, grave consequences on human health emerge in the forms of inherited genetic diseases and cancer. The impact of DNA replication arguably extends far beyond the narrow community of DNA replication researchers and far wide across the nation's health and wealth. Insights into DNA replication at the molecular level provide opportunities to modulate and intervene in rapidly dividing cancer cells. Targeting components of the replication process is potentially a very powerful strategy in the treatment of cancer. A similar approach could also be applied for the development of much needed novel antimicrobials against antibiotic resistant superbugs such as MRSA, Clostridium difficile and Pseudomonas which are major threats of human health. These are long-term strategic targets of many governments in the industrialized research-active world.

Staff working on the proposed research will acquire, apply and develop a range of biological skills (molecular biology, protein chemistry, structural modelling and genetics) professional skills. Such training is highly sought after in industry and in academia. Staff are likely to continue applying these skills well beyond the duration of this research in future employment in either sector. This addresses the BBSRC's emphasis on the importance on people, skills and training. The recent strategic review highlighted a core aim of the BBSRC as "Significantly increasing the economic and social impact of BBSRC-funded research by helping to provide the skilled researchers needed for industrial R&D and academic research."

I am also putting great emphasis on communicating with the public to highlight the importance of basic science.