Nanomedicine Development Centers
Cell Propulsion LabCenter for Cell ControlCenter for Protein Folding MachineryNanomedicine Center for Nucleoprotein MachinesNanotechnology Center for Mechanics in Regenerative MedicineNational Center for Design of Biomimetic NanoconductorsOptical Control of Biological FunctionPhi29 DNA-Packaging Motor for Nanomedicine

Nanomedicine Center for Nucleoprotein Machines

Executive Summary

Nucleoprotein machines manage the business of information storage and transfer inside cells, including DNA replication and repair, RNA synthesis, and protein translation. Our center's science and engineering focus is to develop tools and concepts to better understand nucleoprotein machines. Our central prototype is the non-homologus end joining (NHEJ) complex for repairing DNA double stranded breaks. We are combining bright photostable probes, super-resolution microscopy, and other physical and computational tools to investigate assembly, disassembly, and control of the NHEJ complex. We also hope to engineer new protein machines that can provide genetic fixes for common human diseases. Such machines would be most applicable to diseases that are reasonably common, life-threatening, caused by a single-gene defect, and where correction of the defect in even a fraction of cells would provide clear clinical benefit. Examples of diseases that meet these criteria are hemoglobinopathies, such as sickle cell disease, and triplet-repeat expansion neurodegenerative diseases, such as Huntington's disease.

Introduction

The mammalian cell nucleus is filled with self-organizing, interconnected, nanometer-scale machines that carry out DNA replication, RNA synthesis, RNA transport and DNA repair, and other processes [1]. These nucleoprotein machines are made primarily of protein, and act on nucleic acid substrates. They are complex: synthesis of a typical human mRNA, for example, requires interaction of hundreds of protein and RNA components that perform many processes including initiation, capping, elongation, splicing, polyadenylation, and termination.

To meet the challenges of nanomedicine, we need to first establish the structure-function relationship for each nucleoprotein machine. This includes how the machines assemble and disassemble, as well as their signalling and control mechanisms. Nucleoprotein machines work with a common set of raw materials (nucleotides and polynucleotides), carry out similar elementary steps (nucleotidyl and phosphoryl group transfer), and often have interchangeable components. These similarities suggest that the study of different nucleoprotein machines will reveal common, and generalizable, engineering design principles. However, nucleoprotein machines often do not have a fixed composition. They are dynamic with proteins that associate and dissociate, depending on the specific functional state of the biological process. As a model, we study the nonhomologous end joining (NHEJ) complex that repairs DNA double-strand breaks (DSBs) in mammalian cells. This complex requires a different constellation of factors depending on whether it is removing damaged nucleotides or adding new nucleotides to fill a sequence gap [2]. NHEJ occurs within chromatin domains of roughly 2 million base pairs of DNA and must be able to join ends of virtually any nucleotide sequence. Since each repair complex is unique at any instant in time, it is essential that we study the behavior of single nucleoprotein complexes, rather than population averages, in order to gain insight into their design and function. Our research is inspired by the natural process of V(D)J recombination, which uses a double-strand break repair machine to insert, delete, and alter DNA sequences at specific sites for generating T-cell receptors and immunoglobulins that are critical for defense against bacteria and viruses.

Why the NHEJ Complex?

Initially we are focusing on the nonhomologous end joining (NHEJ) pathway that repairs DNA DSB because:

Figure 1
Figure 1. A model for NHEJ complex formation in mammalian Cells. The Ku70/Ku80 heterodimer forms a hollow ring that preferentially binds to DNA ends. Binding of the Ku protein recruits DNA-PKcs, which forms a complex with the Artemis nuclease. DNAPKcs may tether the ends, while Artemis nucleolytically processes DNA ends prior to joining. The Cernunnos-XLF protein forms complexes with XRCC4, Ligase IV, or XRCC4 and Ligase IV simultaneously. The exact nature of the active complex is currently undefined, but could involve the formation of heteromultimers with XRCC4 or the XRCC4-Ligase IV complex. The final stage of NHEJ is the ligation of DNA ends catalyzed by XRCC4- Ligase IV. Cernunnos-XLF promotes this process in an unknown way.

What do we know about how NHEJ fixes broken DNA?

A working model of the NHEJ function is shown in Figure 1. The Ku protein (composed of Ku70 and Ku80 subunits) carries out initial end recognition. Ku recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which sequesters the DNA ends. The DNA ligase complex (composed of DNA ligase IV, XRCC4, and XLF) then catalyzes phosphodiester bond formation. These six polypeptides are sufficient for the core NHEJ reaction. Mammalian cells that are deficient in any of the core NHEJ components are very sensitive to DSB-inducing agents. Most of the core NHEJ components (except DNA-PKcs) are required for an analogous NHEJ pathway in budding yeast, which has allowed analysis of NHEJ in this genetically tractable model organism.

NDC Aims

To fully understand the design of nucleoprotein machines, it is necessary to characterize the dynamics of the NHEJ complex in living cells. However, visualizing single NHEJ complexes in the nuclei of living cells is far beyond the limits of existing technology, and progress toward this goal requires a multidisciplinary team of biologists, bioengineers, chemists, and computational scientists.

Our first step is to synthesize new optical imaging probes that are, small (4-6 nm) and biocompatible, and then develop new strategies to attach these probes to individual components of the NHEJ complex. After labelling 2, 3 or 4 components, we will visualize its assembly in vitro using single molecule techniques. We are determining the best methods to induce controlled DSBs in vivo and will use optical imaging methods with improved sensitivity and spatial resolution to characterize the assembly and disassembly of the repair nanomachine on a chromatin substrate in living cells. Developing new imaging capabilities will also allow us to measure the dimensions of repair foci as a function of time following DSB induction, and investigate their internal structure and composition using high resolution methods applicable to fixed cells. We will use electron cryo-microscopy, dual contrast probes, and novel sample preparation methods.

Next, we will need to elucidate the signaling-response-feedback-control loops that connect different aspects of the DSB response and develop a mathematical model for quantifying the reaction kinetics, the growth and disappearance of repair foci, and the amplification of DNA damage signals. Simulation of nanomachine formation will permit us to interpret and quantify the experimental data and understand the design of nucleoprotein machines. In other words, we want to be able to characterize and build models that describe the assembly, the disassembly, and the function of the complex that we can test and use to predict how we can control its function.

An important extension of these studies includes work on V(D)J recombination, a mechanism of DNA recombination used by the immune system to generate a diverse assortment of T-cell receptors and immunoglobulins that are critical for antigen recognition and defense against bacteria and viruses. This work is integral to our NDC because V(D)J recombination uses the core NHEJ machine [3].

NDC accomplishments

A major near-term goal of our NDC is to track the assembly and disassembly of single NHEJ complexes in living cells. To achieve this goal, over the last 12 months, we developed methods for labeling NHEJ components in both mammalian and yeast cells. We also have improved our fluorescence, live-cell imaging methods with multi-color capabilities, as well as instrumentation and various techniques for single-particle imaging in electron microscopy. Further, experimental and analytic tools have been developed to characterize the dynamic properties of NHEJ proteins in the absence of DSBs, especially the low-affinity binding reactions.

Labeling NHEJ components

Several independent efforts to tag individual components are underway. Use of one or a combination of these will permit visualizing NHEJ complex dynamics.

Figure 2
Figure 2: Fluorescent protein tagging strategies. (A) Spectral characteristics of four fluorescent proteins that can be used for visualizing components of the nucleoprotein machine. (B) Examples of fluorescently tagged NHEJ proteins. Proteins were expressed transiently in HeLa cells under control of a CMV promoter. Images were collected by multiphoton scanning confocal microscopy and have been merged with a differential interference contrast image of the same field. Adapted from Ref 5.

NHEJ Function

We are beginning to examine the function of tagged NHEJ components by testing their ability to complement NHEJ function in mutant cell lines. So far, we have verified the ability of fluorescently tagged proteins to migrate to sites of DNA damage. Although eventually we want to track movement of the NHEJ components to enzymatically induced DSBs at single sites in living cells, as an interim substitute, we used a system where DNA damage was induced using a multiphoton confocal microscope.

Imaging NHEJ in yeast

Although not identical to the mammalian NHEJ complex function, many components in yeast are identical and this more simplistic, genetically modifiable system, allows us to gain insight into the best approaches and most appropriate studies in mammalian tissues. We constructed a series of yeast strains that harbor NHEJ genes fused to a fluorescent protein tag focusing on the genes that constitute the core complex of the NHEJ reaction.

All GFP-tagged strains showed fluorescence signal that was restricted to the nucleus of the cells, as expected. The strains were tested for their ability to generate repair foci upon exposure to the DNA damaging agent doxorubicin. Doxorubicin generates DNA double-strand breaks by inhibiting DNA topoisomerase II. Two strains (RAD27-GFP and YKU80) presented discrete foci after exposure to doxorubicin and will therefore be useful in functional studies.

Viewing Living Cells

In addition to developing imaging probes, tagging strategies, and fluorescent-protein labeled NHEJ components, we devote considerable effort to optimizing live cell imaging using multiple fluorescent proteins. Using a stable osteosarcoma cell line, we evaluated the recruitment of multiple fluorescent proteins to a specific nuclear site in living cells. This study lays the groundwork for future studies aimed at understanding the assembly/disassembly of the NHEJ DNA repair machinery at specific sites in the interphase nucleus.

Our NDC recently installed two DeltaVision (Applied Precision) live-cell imaging systems that are equipped with simultaneous multi-color imaging capability, two lasers for FRAP and photoactivation, and a module for TIRF (Total Internal Reflection Fluorescence) studies. These fluorescence microscopes provide reduced photobleaching and better software for image analysis, thus significantly increasing the sensitive and signal-to-noise ratio in visualizing single nucleoprotein machines in living cells.

Live cell imaging of single nucleoprotein machines is very challenging. With fluorescent protein (FP) tagging approach, we will need to visualize a small number (5-10) FPs co-localized in a living cell of which low signal level and photobleaching are major issues. We are exploring the possibility of acquiring a new live-cell imaging system that has minimal photobleaching and enhanced sensitivity. Using quantum dots, large size, interference with assembly, and signal-to-noise ratio are all major issues. We are therefore developing a FRET-based alternative tagging strategy to mitigate this issue. To validate the protein-tagging approaches, we will demonstrate the NHEJ formation and functionality using in vitro methods.

Intracellular mobility of NHEJ proteins

We recognize the extremely complex environment of the nucleus which has a viscosity considerably higher than water and which no longer is viewed simply as a dilute, fluid-filled compartment. Consequently, we have begun to characterize the non-specific interactions and dynamic properties of NHEJ proteins in the absence of DSBs, we analyzed the protein mobility by fluorescence recovery after photobleaching (FRAP). To do so, we generated expression plasmids encoding GFP-fusions of human Ku86 and Ku70 and transiently transfected human HeLa cells to measure the protein mobility of GFP-Ku70 and GFP-Ku86 24 h using FRAP. Assuming that the interaction with chromatin takes place much faster than diffusion, we determined that GFP-Ku86 could be fitted to a simple model and obtained the corresponding diffusion coefficient that was markedly less than expected given known values of intracellular viscosity. We continue to explore the determinants of this reduced mobility.

Future Applications

The tools and novel approaches developed in this NDC can be readily extended for a broad range of applications in both basic biological studies and translational research of diseases. With further development, our tool set can be expanded to study the RNA synthesis and mRNA splicing machinery by selecting and tagging a subset of the components (>100) contained in these nanomachines. In particular, the imaging methods and computational tools can be generalized to the studies of almost any multiprotein molecular machines inside living cells. The protein tagging strategies and imaging probes can be used for studying signal transduction pathways, gene regulation pathways, protein-nucleic acid interactions, disease analysis and detection, and cell-based assays for the development of new drugs.

The design of nucleoprotein machines in living cells has been optimized by nature over more than a billion years. These machines can realize their functions with astonishing precision, efficiency, and robustness. We envision that eventually we will be able to: (1) engineer nanomachines to physically and topologically isolate their DNA substrate; (2) design different nanomachines that have interchangeable parts, for example, small protein assemblies that execute the same preset series of actions; (3) utilize repeating polymeric structures of DNA and RNA to form natural biological amplifiers, to allow a signal initiating at a single site to propagate via chromatin modification, providing a long dock for signaling proteins that arrive, undergo modification, and depart to propagate the signal three-dimensionally.

A long-term goal of our NDC is to adapt DNA repair nanomachines for therapeutic benefits by precisely modifying the information stored in DNA and RNA, thus providing genetic cures for common human diseases. Nearly all human diseases have a genetic component: cancer reflects age-dependent acquisition of somatic mutations, cardiovascular disease and diabetes risk reflect inherited metabolic traits, and hemoglobinopathies, lysosomal storage diseases, and inborn errors of metabolism reflect point mutations. Modern medicine - allopathic medicine - focuses on treating symptoms, commonly through small-molecule enzyme inhibitors and receptor agonists/antagonists and does not address underlying genetic causes. Consistent with the vision of the Nanomedicine Roadmap Initiative, we anticipate that, in the future, the allopathic model can be replaced by therapies that directly modify information contained in DNA and RNA. One possibility is to treat some of the familial breast and colorectal cancers with known failures of DNA surveillance repair, or the leukemias (e.g., CML) with known translocations, by utilizing the DNA damage repair machines. It is also possible to treat neurodegenerative triplet repeat expansion diseases, such as Huntington's disease, by correcting the genetic defects using re-engineered nucleoprotein machines.

Specifically, we conceptualize the medical application of our nanomedicine efforts, or our Pathway to Medicine (PtM), in achieving this long-term goal in terms of three elements: device, delivery, and a specific disease target [5]. We have identified candidates for each. For the device, one approach that we are exploring capitalizes on a recent discovery that the recombination activator gene (Rag) proteins, which normally initiate a specialized V(D)J recombination reaction in developing lymphocytes, can be re-engineered to promote site-specific homologous recombination [3, 6]. In principle, this can be achieved for any gene in any cell. For the delivery system, we will develop an approach based on nanoparticles as a carrier, which could have multiple functions, including delivery, targeting and release functions. By tailoring the size, surface coating and targeting ligand of the nanoparticle, optimal performance in delivery will be achieved. For the disease, we have tentatively selected sickle cell disease and Huntington disease as our initial disease models. These diseases are fairly common, and life-threatening. Importantly, each is caused by a single-gene defect, and the disease mechanism is such that 100% efficient correction of the disease genes should not be required for clinical efficacy. Inspired by the NHEJ and V(D)J recombination pathways, we will harness DNA repair nanomachines to alter, insert, delete sequence as a novel way to cure human diseases.

References

Background

  1. Spector DL: The dynamics of chromosome organization and gene regulation. Annu Rev Biochem, 72, 573-608 (2003).
  2. Sekiguchi JM, Ferguson DO: DNA double-strand break repair: a relentless hunt uncovers new prey. Cell, 124, 260-262 (2006).
  3. Lee GS, Neiditch MB, Salus SS, Roth DB: RAG proteins shepherd double-strand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination. Cell, 117, 171-184 (2004).
  4. Howarth M, Takao K, Hayashi Y, Ting AY: Targeting quantum dots to surface proteins in living cells with biotin ligase. Proc Natl Acad Sci U S A, 102, 7583-7588 (2005).

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