DNARepairMan

An innovative training network aimed at understanding the regulated assembly of molecular machines for DNA repair through design and implementation of integrative technology

Project description

The European Training Network DNARepairMan aims to provide the next generation of young scientists with a unique and innovative interdisciplinary training in cutting-edge biophysical research methodologies to address central questions in biology with relevance for human health. This is motivated by the understanding that the future development of novel targeted therapies and antimicrobials will rely precisely on understanding the molecular and mechanistic details underlying disease.

Our focus on the process of DNA repair arises from its essential role in maintaining genome stability. In this process multiple proteins collaborate as large molecular machines to detect and repair very rare errors that occur in DNA. These evolutionarily-conserved processes are of fundamental importance, and there is an increasing awareness that targeting of DNA repair pathways could also be useful in the development of personalized anticancer therapies and new antimicrobials.

The importance of mechanistic understanding of DNA repair pathways has further been recognized by the award of the 2015 Nobel Chemistry Prize to three pioneers in the field of DNA repair. Nevertheless, even today important questions remain as to how the relevant enzyme complexes collaborate and assemble, despite stochasticity and fluctuations, to robustly coordinate repair. Recent developments of highly sophisticated biophysical and structural technologies now enable the analysis of these transient systems at unprecedented spatial and temporal resolution. To capitalize on the virtuous cycle between new technologies, new questions and new insights we therefore propose the DNARepairMan training network.

Within the DNARepairMan Network we aim to train young researchers at the highest level of rigor and excellence by providing them with both the theoretical and quantitative tools required to address central questions of contemporary biology regarding the mode of action of dynamic molecular machineries, but also by involving them in the development and implementation of innovative experimental methods.

This Training Network uniquely brings together biologists, chemists, and physicists around a common research question: what are the statistical properties and molecular mechanisms of the switches and motors involved in two canonical DNA repair pathways? Using innovative methodologies to trap and visualize transient reaction states will allow for detailed definition of the relevant molecular mechanisms. These studies will provide insight into the basic chemical and physical principles that govern correct timing and order of events within complicated molecular processes.

The different participants bring together highly complementary research programs each situated at the forefront of their respective research fields, providing a cooperative and creative atmosphere that will allow young researchers to grow and gain expertise by training-through-research. All participants will share their experience, knowledge, and state-of-the-art tools and techniques, creating an exceptional multidisciplinary training network for young researchers. These students will be thoroughly trained in their own discipline, but will also receive in-depth interdisciplinary training by exposure to complementary approaches present in the network addressing the same research question.

Furthermore, by incorporating four technology-driven companies, including a business development unit, the consortium contributes to and benefits from improvements in and transfer of methodology, instrumentation and product development from academia to industry and vice versa. Finally, through direct contacts with the creative sector, young researchers will integrate, translate and disseminate their research findings and new standards to the larger scientific community, to industry and to society.

Objectives

The overall research aim of the project is to unravel the mechanistic details of the regulated assembly of molecular machines for DNA repair. Academic and industrial laboratories will develop new reagents, assays, technology and software to analyse fundamental chemical and physical principles underlying the molecular organization of two critical DNA repair pathways: DNA mismatch repair and transcription-coupled nucleotide excision repair. The objectives of the research program address the different levels of lesion recognition, signaling complex assembly and recruitment and regulation of DNA unwinding within the two pathways,

  1. to establish the mechanism of lesion recognition complex formation,
  2. to determine the composition and structure of the helicase recruitment complexes,
  3. to characterize the catalytic properties of the unwinding complexes,
  4. to understand the regulation of their activity,
  5. to establish the link between DNA repair and replication.

 

Overview of the research programme

In the DNARepairMan Network we study mechanistic details of the communication between molecular machines within two critical DNA repair pathways using state-of-the-art technologies. The ESRs will be immersed in a multidisciplinary training environment in fundamental life sciences and obtain insight into the role of basic chemical and physical principles underlying molecular steps in complex yet integrated molecular processes. As the research relies on cutting-edge technologies, development of new approaches will be an integral part of the individual research projects. Thanks to the close collaboration between academia and industry, the early-stage researchers (ESRs) will further learn how technology development drives innovation in research and how basic research questions can drive innovative technology into marketplace developments.

The repair of DNA damage is a critical step in maintenance of genome integrity. Errors in DNA repair pathways generate mutations and ultimately result in ageing and cancer in humans. A better understanding of these pathways will result in better diagnosis and ultimately, improved treatment of cancer. The processes involved are executed by complex molecular machineries that execute a carefully choreographed set of activation steps. In this project we make use of extremely innovative and complementary approaches that will allow an integrated vision of the steps in DNA mismatch repair (MMR) and transcription coupled repair (TCR).

MMR is a critical DNA repair system that guards the genome against errors that occur during replication; it also plays important roles in DNA damage signaling and recombinationi. The basic MMR machinery is highly conserved from bacteria to humans and dysfunctions in the MMR machinery lead to a predisposition to familial (colon) cancer in humans, known as Lynch syndrome. MMR involves a cascade of ATPases that read out the mismatch, and ultimately ensure the specific repair of the newly synthesized strand. This process is initiated when the homodimeric MutS protein or its eukaryotic heterodimeric homolog binds to a mismatch or looped out base. MutS then releases the mismatch and forms a stable ATP-dependent clamp that can slide on DNA and that is able to activate MutL, the next ATPase in the cascade. This activated state of homodimeric MutL, or its heterodimeric eukaryotic homolog, is required to activate a nuclease that incises only the newly synthesized DNA strand. In E.coli, this nuclease is MutH, which nicks the unmethylated strand at a temporarily hemi-methylated GATC site. The helicase UvrD is then loaded at the site of incision to unwind the DNA, exonucleases will digest the daughter strand and the gap thus generated is filled in by polymerase and sealed by ligase. In eukaryotes, the nuclease activity is intrinsic to the heterodimeric MutL homologs, which becomes activated in a strand-specific manner by the replication processivity clamp PCNA. Although the basic steps in MMR are reasonably well understood, the manner in which these molecular machines activate each other and how they communicate along the DNA from the mismatch to the point that signals the new strand (a hemimethylated GATC in E.coli) remains an essential yet unresolved question. It is noteworthy that even the temporal composition and stoichiometry of the relevant active complexes is still highly debatedii. New insights regarding this machinery are required to better understand its canonical activity as well as its more elusive roles in signaling and recombination. Importantly, exploitation of the lack of MMR has potential in selected synthetic lethal drug combinations that target cancer cellsiii, in a manner similar to the successful combination of BRCA1 deficiency and PARP inhibitioniv.

Nucleotide excision repair (NER) targets bulky DNA lesions, such as UV photoproducts and alkylated bases, and occurs via at least two major subpathwaysv . The "global" NER pathway (GGR) can act upon lesions anywhere within the genome, and the transcription-coupled NER pathway (TCR) acts only upon lesions in the template strand of transcribed genes. Loss of NER in prokaryotes or eukaryotes leads to UV-sensitivity, and in bacteria, proteins of the TCR pathway are involved in promoting mutagenesis that can lead to antibiotic resistancevi. In bacteria, GGR involves a multi-step damage-search and recognition process, performed by the repair proteins UvrA and UvrB, followed by dual-incision on either side of the lesion by the nuclease UvrC, removal of the short (12-13 nt) damage-bearing oligonucleotide by the helicase UvrD, and then repair patch synthesis by DNA polymerase I and DNA ligase. TCR requires RNA polymerase (RNAP) and a transcription-repair coupling factor, Mfd, in addition to all of the proteins that are required for GGR. Mfd-mediated TCR is faster than GGR, and hence lesions in the template strand of active genes are repaired more quickly than lesions elsewhere in the genome. The protein components of the TCR pathway are well characterized, but the mechanisms by which they interact to locate DNA damage and initiate repair remain poorly understood.

MMR and NER/TCR have interesting mechanistic similarities, and also share a key protein component. In both cases the lesion in the DNA (a replication error or a bulky DNA lesion) is first bound by lesion recognition protein(s) (MutS, RNAP) that subsequently recruit adaptor proteins (MutL or Mfd/UvrA/UvrB). ATP-dependent rearrangements then lead to recruitment of a nuclease to perform strand incision (MutH or UvrC) and a helicase to unwind the DNA (in both pathways this is UvrD). Outstanding mechanistic questions focus on this last step of DNA unwinding in both pathways: how is UvrD recruited, how is it activated, how does it know in which direction it has to unwind in order to remove the lesion, and how does it know when to stop? There are also similarities between the ATP-dependent movement of UvrD from a distant nick site to the lesion in MMR and the ATP-dependent movement of Mfd from a paused RNAP towards the lesion in TCR. The overall research question for this training Network is thus: how are these molecular machines properly recruited and how is their activity properly regulated during DNA repair?

 

 

iModrich, P. (2006) The Journal of biological chemistry, 281, 30305-30309 (PubMed); Jiricny, J. (2013) Cold Spring Harbor perspectives in biology, 5, a012633. (PubMed)
iiElez, M. et al. (2012) Nucleic acids research, 40, 3929-3938 (PubMed); Lee, J.B. et al. (2014). DNA repair, 20, 82-93. (PubMed)
iiiMartin, S.A. et al. (2010) Clinical cancer research 16, 5107-5113. (PubMed)
ivFarmer, H. et al. (2005) Nature, 434, 917-921. (PubMed)
vKisker, C. et al. (2013) Cold Spring Harbor perspectives in biology, 5, a012591. (PubMed)
viHan, J. et al. (2008) PLoS pathogens, 4, e1000083.(PubMed)