"While DNA could be claimed to be both simple and elegant, it must be remembered that DNA almost certainly originated fairly close to the origin of life when things were necessarily simple or they would not have got going.."
DNA repair pathways maintain human health by preventing the accumulation of mutations in DNA. Mutations arise by
- replication of bases that have been chemically altered by intrinsic or extrinsic factors, or
- replication of bases inserted erroneously in a previous round of replication.
Both chemically altered and mismatched bases we refer as DNA damage. If left unchecked, unrepaired DNA damage pre-disposes us to cancer and developmental defects. Accordingly, inherited or somatic mutations in DNA repair proteins are strongly associated with human disease. Moreover, DNA repair pathways are drug targets because they help tumor cells to tolerate chemotherapeutics, and enable bacteria to become resistant to antibiotics.
An understanding of the structures and mechanisms of the molecular components of specific DNA repair pathways is key to identifying and exploiting targets for pharmaceutical intervention, but to predict the outcome of such intervention on the overall repair capacity of the cell it is necessary to also understand the interconnected web of functional and regulatory interactions that links different pathways together.
Recent advances in cryo-EM, single molecule analysis, structural proteomics and kinetic studies using fluorescent probes, together with longer-established biochemical and genetic approaches, now make it possible to monitor the formation and disassembly of molecular complexes at an unprecedented scale and level of precision.
In RepState we bring together expertise in these areas to ask how an interconnected network of DNA repair enzymes switches between multiple different states to protect DNA from the effects of nucleotide mis-incorporation (mismatch repair (MMR)) and bulky DNA damage (nucleotide excision repair (NER) and transcription-coupled NER (TC-NER)).
DNA repair by individual proteins and linear pathways.
- the complexity of DNA repair in the cellular environment,
- the extensive crosstalk that we know exists between different repair pathways and
- that the proteins involved almost always act in macromolecular complexes as opposed to in isolation.
The overarching aim of our network is to acquire and synthesize new mechanistic insights into DNA repair proteins to provide a systems level description of DNA repair.
Both MMR and the various forms of NER can be regarded as a “black box” that converts different types of damaged DNA into undamaged DNA. Each contains common sub-steps such as specific recognition of the lesion leading to activation of the appropriate pathway and so forth, but they achieve these steps by distinct mechanisms. In both cases DNA repair is orchestrated by a network of DNA repair proteins, which bind to damaged DNA and access ATP to provide energy for repair, and they are functionally connected by a common enzyme component (a motor called UvrD), and via the cell’s ATP pool.
Inspired by concepts from computer programming, we consider these DNA repair pathways at multiple levels as “state machines”, or devices that exist in a set number of stable conditions depending on their previous conditions and the status of various inputs.
- At one level the complete network of DNA repair proteins and interacting factors within the cell can be viewed as a state machine. A large but finite combination of complexes and enzymatic activities are possible, and the state of the machine depends on inputs such as DNA damage and metabolic state of the cell.
- The second level at which the state machine analogy applies is for individual protein components, which convert between different states in response to interaction with partner proteins, DNA, ATP etc.
The activity of each individual protein at any moment is determined by the combination of binding partners as well as by the order in which binding of these partners has occurred.
This figure illustrates this concept for the UvrD protein, which participates in multiple repair pathways and switches between inactive and (multiple) active states by interaction with other cellular components.
Ultimately, the various protein state machines act together to integrate different DNA damage signals and the system responds by activating the right proteins at the right time to access energy from the ATP pool and drive productive DNA repair events.
Our vision is to describe the repair machinery of the cell by defining all possible physical states of each component and the probability of reversible transitions between them.
This requires a detailed understanding of the architecture of the free repair proteins, the complexes they form with each other, and with DNA and ATP, the different possible conformations adopted by such complexes, and the kinetics of the state transitions. Building upon the advances from mismatch2model and DNARepairMan, RepState is initiating this next step to study the mechanistic principles underlying the activities and interactions of multi-protein repair machines requires a unique and interdisciplinary skill set. RepState is a consortium consisting of cell biologists, biochemists, biophysicists, and structural biologists, whose expertise is being integrated to gain understanding of how these complexes assemble and relay signals in a coordinated manner.
RepState aims to create a Doctoral Training Network focused on unraveling the molecular details of the mode of action of DNA Repair State Machines that are crucial for maintaining genome stability. RepState is doimg so by developing new technologies with high temporal and spatial resolution to study structure and function of DNA Repair State Machines for the two crucial, connected DNA repair pathways MMR and NER.
To understand how the state machines coordinate reaction sub-steps, RepState has formulated five research objectives that reflect successive reaction steps and their corresponding state machines in the MMR, NER and TC-NER pathways:
Uncover the mechanism of activation of lesion recognition state machines
Dissect how this activation regulates multiple downstream actions on DNA
Decipher how DNA incision is activated
Decipher how DNA unwinding is activated
Unravel how the coordinated function of multiple state machines results in efficient DNA repair
RepState creates a unique training environment in skills and experiences
RepState is training a new generation of interdisciplinary biochemists and biophysicists to appreciate
- how new technologies can be combined to generate novel insights and
- how the fundamental physical and chemical properties of molecules relate to their behavior within a living cell.
The researchers are also learning to create and benchmark highly pure and homogeneous reagents and operate cutting-edge technologies. Furthermore, the students are exploring new way and means to communicate their results and the importance of fundamental research in general to society.
This combination of expertise is not provided for by existing educational programs. Our network would therefore create a unique training environment in skills and experiences that are both highly valued and in short supply within the expanding biosciences sector.