Molecular hand-off mechanisms during lagging strand replication

Lead Research Organisation: University of Nottingham
Department Name: Sch of Chemistry

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description We have studied extensively the essential bacterial DNA replication protein SSB and have defined the structural state of its intrinsically disordered C-terminal domain. The generally accepted, albeit speculative, mechanistic model in the field postulates that binding of ssDNA to the OB core induces the flexible, undefined C-terminal arms to expand outwards encouraging functional interactions with partner proteins. We showed that the opposite is true. Combined small-angle scattering with X-rays and neutrons coupled to coarse-grained modeling reveal that the intrinsically disordered C-terminal arms are relatively collapsed around the tetrameric OB core and collapse further upon ssDNA binding. This implies a mechanism of action, in which the disordered C-terminal domain collapse traps the ssDNA and pulls functional partners onto the ssDNA. We have also explained how to produce a B. subtilis SSB probe that exhibits 9-fold fluorescence increase upon binding to single stranded DNA and can be used in all related gram positive firmicutes which employ drastically different DNA replication and repair systems than the widely studied Escherichia coli. The materials to produce the B. subtilis SSB probe are commercially available, so our methodology is widely available unlike previously published methods for the E. coli SSB. In additional studies with Neisseria meningitidis we discovered a novel mechanism by which pathogens use the twitching motility mode of the Tfp machinery for sensing and importing host elicitors, aligning with the inflamed environment and switching to the virulent state.
Exploitation Route We have provided some tools to assay in real time bacterial DNA replication and revised a widely accepted model for SSB action which turns out to be untrue. We identified and characterized the mechanistic details of the putative replicative helicase (CD3657), helicase-loader ATPase (CD3654) and primase (CD1454) of C. difficile, and reconstitute helicase and primase activities in vitro We demonstrated a direct and ATP-dependent interaction between the helicase loader and the helicase. Furthermore, we found that helicase activity is dependent on the presence of primase in vitro The inherent trinucleotide specificity of primase is determined by a single lysine residue and is similar to the primase of the extreme thermophile Aquifex aeolicus. However, the presence of helicase allows more efficient de novo synthesis of RNA primers from non-preferred trinucleotides. Thus, loader-helicase-primase interactions, which crucially mediate helicase loading and activation during DNA replication in all organisms, differ critically in C. difficile from that of the well-studied Gram-positive Bacillus subtilis model.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description We demonstrate a direct and ATP-dependent interaction between the helicase loader and the helicase. Furthermore, we find that helicase activity is dependent on the presence of primase in vitro The inherent trinucleotide specificity of primase is determined by a single lysine residue and is similar to the primase of the extreme thermophile Aquifex aeolicus. However, the presence of helicase allows more efficient de novo synthesis of RNA primers from non-preferred trinucleotides. Thus, loader-helicase-primase interactions, which crucially mediate helicase loading and activation during DNA replication in all organisms, differ critically in C. difficile from that of the well-studied Gram-positive Bacillus subtilis model. The bacterial single-stranded DNA (ssDNA) binding protein SSB is a strictly conserved and essential protein involved in diverse functions of DNA metabolism, including replication and repair. SSB comprises a well-characterized tetrameric core of N-terminal oligonucleotide binding OB folds that bind ssDNA and four intrinsically disordered C-terminal domains of unknown structure that interact with partner proteins. The generally accepted, albeit speculative, mechanistic model in the field postulates that binding of ssDNA to the OB core induces the flexible, undefined C-terminal arms to expand outwards encouraging functional interactions with partner proteins. In this structural study, we show that the opposite is true. Combined small-angle scattering with X-rays and neutrons coupled to coarse-grained modeling reveal that the intrinsically disordered C-terminal arms are relatively collapsed around the tetrameric OB core and collapse further upon ssDNA binding. This implies a mechanism of action, in which the disordered C-terminal domain collapse traps the ssDNA and pulls functional partners onto the ssDNA. In most cases its interactions with partner proteins is species-specific and for this reason, knowing how to produce and use cognate reagentless SSB biosensors in different bacteria is critical. Here we explain how to produce a B. subtilis SSB probe that exhibits 9-fold fluorescence increase upon binding to single stranded DNA and can be used in all related gram positive firmicutes which employ drastically different DNA replication and repair systems than the widely studied Escherichia coli. The materials to produce the B. subtilis SSB probe are commercially available, so the methodology described here is widely available unlike previously published methods for the E. coli SSB. We have also revealed that important protein-protein interactions regulate the functional properties of the B. subtilis replisome. More specifically we have shown that (i) the polymerase activity of DnaE is essential for both the initiation and elongation stages of DNA replication, (ii) its error rate varies inversely with PolC concentration, and (iii) its misincorporations are corrected by the mismatch repair system post-replication. We also found that the error rates in cells encoding mutator forms of both PolC and DnaE are significantly higher (up to 15-fold) than in PolC mutants (iv) the polymerase activity of DnaE is considerably stimulated by DnaN, SSB and PolC, (v) its error-prone activity is strongly inhibited by DnaN, and (vi) its errors are proofread by the 3' > 5' exonuclease activity of PolC in a stable template-DnaE-PolC complex. Collectively our data will aid the pharmaceutical industry in the development of new antimicrobials that are much needed in this era of aggressively emerging antimicrobial resistance. Our new functional discoveries will offer potential new targets against antimicrobial resistance. Companies could develop specific drugs that target the essential structural/functional interactions discovered in our research.
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Title Reagentless SSB biosensor 
Description We have created a fluorescently labelled reagentless SSB biosensor that can be used to assay bacterial DNA replictaion reactions in real time in vitro 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact This reagent can be used by the bacterial replication community to assay replication events real time and to reconstitute replication systems in vitro 
URL http://www.ncbi.nlm.nih.gov/pubmed/24953846
 
Description Collaboration with Dr. Laurent Janniere (expert in Bacillus subtilis genetics and DNA replication) 
Organisation French National Institute of Agricultural Research
Country France 
Sector Public 
PI Contribution We have verified genetic data obtained by our collaborator and we have revealed functional protein interactions related to the Bacillus subtilis DNA replication system.
Collaborator Contribution Our partner contributed genetic studies using his extensive genetics tools in Bacillus subtilis to reveal potential protein links and interactions that regulate the functions of replication proteins.
Impact (1) Interactions of the Bacillus subtilis DnaE polymerase with replisomal proteins modulate its activity and fidelity. Paschalis V, Le Chatelier E, Green M, Képès F, Soultanas P, Janniere L. Open Biol. 2017 Sep;7(9). pii: 170146. doi: 10.1098/rsob.170146.
Start Year 2015
 
Description International and national collaborations 
Organisation Rutherford Appleton Laboratory
Country United Kingdom 
Sector Public 
PI Contribution We studied the intrinsic disorder of the essential bacterial replication/repair protein SSB. My team was the main contributors.
Collaborator Contribution Emre Brookes provided some theoretical calculations and compouting assistance. David Scott and louise Hatter providing assistance with SAXS and SANS experiments at Harwell.
Impact Published paper Defining the Intrinsically Disordered C-Terminal Domain of SSB Reveals DNA-Mediated Compaction. Green M, Hatter L, Brookes E, Soultanas P, Scott DJ. J Mol Biol. 2016 Jan 29;428(2 Pt A):357-64. doi: 10.1016/j.jmb.2015.12.007.
Start Year 2014
 
Description International and national collaborations 
Organisation University of Texas at San Antonio
Country United States 
Sector Academic/University 
PI Contribution We studied the intrinsic disorder of the essential bacterial replication/repair protein SSB. My team was the main contributors.
Collaborator Contribution Emre Brookes provided some theoretical calculations and compouting assistance. David Scott and louise Hatter providing assistance with SAXS and SANS experiments at Harwell.
Impact Published paper Defining the Intrinsically Disordered C-Terminal Domain of SSB Reveals DNA-Mediated Compaction. Green M, Hatter L, Brookes E, Soultanas P, Scott DJ. J Mol Biol. 2016 Jan 29;428(2 Pt A):357-64. doi: 10.1016/j.jmb.2015.12.007.
Start Year 2014
 
Description National collaboration with David Scott (Nottingham) and Mark Dillingham (Bristol) 
Organisation University of Bristol
Department School of Biochemistry Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution We worked in close collaboration to engineer a reagentless SSB biosensor. My group was the main contributors with David Scott and Mrk Dillingham providing supportive experimental expertise.
Collaborator Contribution Mark Dillingham did fluorescence helicase assays to support the use of the SSB biosensor to detect real time DNA replication. David SCott helped us with some data interpretations.
Impact Paper published. Engineering a reagentless biosensor for single-stranded DNA to measure real-time helicase activity in Bacillus. Green M, Gilhooly NS, Abedeen S, Scott DJ, Dillingham MS, Soultanas P. Biosens Bioelectron. 2014 Nov 15;61:579-86. doi: 10.1016/j.bios.2014.06.011
Start Year 2013
 
Description National collaboration with David Scott (Nottingham) and Mark Dillingham (Bristol) 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We worked in close collaboration to engineer a reagentless SSB biosensor. My group was the main contributors with David Scott and Mrk Dillingham providing supportive experimental expertise.
Collaborator Contribution Mark Dillingham did fluorescence helicase assays to support the use of the SSB biosensor to detect real time DNA replication. David SCott helped us with some data interpretations.
Impact Paper published. Engineering a reagentless biosensor for single-stranded DNA to measure real-time helicase activity in Bacillus. Green M, Gilhooly NS, Abedeen S, Scott DJ, Dillingham MS, Soultanas P. Biosens Bioelectron. 2014 Nov 15;61:579-86. doi: 10.1016/j.bios.2014.06.011
Start Year 2013