Research in the Dynan laboratory

Cellular systems for the repair of DNA double-strand breaks

Our laboratory's current interests are in the areas of ionizing radiation and DNA repair. Humans are exposed to ionizing radiation from both natural and artificial sources.  Over the last several decades, average radiation exposure in the US has approximately doubled, in part due to the introduction of advanced medical imaging technologies.  Ionizing radiation, in the form of high-energy photons and other particles, deposits energy along discrete, nanometer-scale tracks. This pattern of energy deposition elicits double-strand breaks and other complex, difficult-to-repair DNA lesions. These lead to tissue damage, cancer, and likely premature aging. Our laboratory studies how human cells and model organisms respond to and repair this damage. Our goals are to obtain a better understanding of the fundamental biological process of DNA repair and to use this knowledge to manipulate the DNA repair machinery for therapeutic benefit.

Within this general framework, the laboratory is best known for its use of biochemical approaches to gain insight into the mechanism of nonhomologous end-joining (NHEJ), which is the default pathway for repair of DNA double-strand breaks in humans and other vertebrates. The NHEJ repair system works via direct ligation of broken DNA ends. This system is essential in humans, where it helps maintain genome stability and protects against cancer and other diseases. The NHEJ repair system is also critical for development of the adaptive immune system, which requires repair of programmed DNA breaks.

Our laboratory's early work in this system provided insight into the initial steps of NHEJ. We showed that Ku protein, which binds avidly to DNA ends, recruits the DNA-dependent protein kinase catalytic subunit into an enzymatically active complex. Using cross-linking approaches, we demonstrated that Ku translocates inward from the broken DNA ends, placing the catalytic subunit in direct contact with DNA termini. The catalytic subunit, which is capable of sensing the structure of the DNA end, recruits additional proteins and coordinates subsequent events in the repair process.

To better understand the mechanism of NHEJ, the laboratory adopted the same conceptual approach that led to the identification of Sp1 as a eukaryotic transcription factor: we fractionated cellular extracts and tested these in a cell-free system for their ability to stimulate a complex biochemical reaction, in this case, the covalent joining of two DNA ends. This approach, which we refer to as “biochemical complementation,” led to the identification of two novel repair factors (proteins known as PSF and p54nrb). These factors form a heterodimer that has the surprising property of binding both DNA and RNA, thus providing a novel link between pathways of DNA repair and RNA synthesis.  We are currently investigating biological and biochemical function of the complex using a combination of in vitro and in vivo studies. These include as-yet-unpublished studies of a knockout mouse (created in the laboratory) lacking one of the two subunits of the complex. The laboratory’s work related to the NHEJ pathway of DNA repair has provided projects for more than 15 graduate students and postdoctoral fellows since 1995.

With the move to Emory University in July 2012, one of our goals is to expand the scope and impact of our studies of the basic science of DNA repair. For example, our work to date has led to specific and testable hypotheses about the role of individual repair factors in promoting the pairing, or synapsis, of DNA ends. We hope to test these hypotheses using structural, biophysical, and single-molecule approaches. Emory University is particularly known for existing and emerging strengths in structural biology, advanced microscopy, sequencing, and bioinformatics. The environment will be ideal for taking our studies of nonhomologous end joining to a deeper level of analysis.

Broader effects of radiation exposure, including high charge and energy particle exposure.

Our work on the repair of radiation-induced DNA damage led to an interest in studying the broader radiation response in tissues and organisms. In addition to DNA damage, radiation exposure elicits changes in gene expression, metabolism, and inflammatory signaling. This broader response is particularly important when there is a temporal or spatial separation between the irradiation and its biological effects. An example is when an embryo receives the exposure and the effects are seen in differentiated adult tissues. In other words, the effects are seen in the distant progeny of the cells that were originally exposed. Another example is when exposure involves low, environmentally relevant doses of radiation, where not every cell in the organism is traversed by a radiation track. In such instances, some of the effects of exposure occur as the result of communication between cells that are directly hit by the radiation and their unhit neighbors.

We established two distinct systems to study the broader radiation response in tissues and organisms. One is a teleost (fish) embryo model. We have used both the zebrafish (Danio rerio) and the Japanese medaka fish (Oryzias latipes). Both models have well-developed genetics and offer the advantage of allowing the analysis of large numbers of individuals at relatively low cost. Notably, medaka is a hardier species that is more amenable to certain experiments using high-energy particle radiation. As part of our work with the fish models, we cloned and characterized the zebrafish Ku genes and demonstrated their role in protection of the embryo from radiation effects. We also developed an approach for quantifying radiation effects in the embryo using multiphoton confocal imaging to identify apoptotic cells and reconstruct their positions in three dimensions. In our most recent studies, we have shown that a single, low- dose, exposure provided during embryonic life, induces profound changes in the pattern of gene expression in the progeny of the cells that were originally irradiated that persist for months after exposure.  We hypothesize that one effect of exposure in early life is premature aging.  To test this, we are currently conducting a lifetime study using large cohorts of medaka fish that have been exposed to gamma-ray or particle beam radiation during early development.

We also use mice and human cells for studying the broader radiation response, as part of a project that is based within a NASA Specialized Center of Research (NSCOR) that was established at Emory University in 2011. We hypothesize that a history of radiation exposure compromises the ability of cells to respond to subsequent DNA breaks, either by perturbing the pattern of micro-RNA expression or through other indirect effects. The Center’s work is focused particularly on high charge and energy (HZE) particle radiation, which, in contrast to more familiar X-rays and gamma rays, deposits large amounts of energy along relatively few radiation tracks. HZE particle radiation permeates the cosmos and is of particular concern to air and space crews traveling beyond the protection of the earth’s magnetic field. Particle beam radiation also forms the basis for a new type of radiotherapy that uses energetic ions, such as carbon nuclei, for tumor control. Our laboratory’s move consolidates all four of the NASA Center investigators on the Emory University campus.

With the adoption of a mammalian system and a focus on altered regulation of repair pathways, our studies of the broader response to radiation will move closer to our studies of DNA repair both intellectually and methodologically.

Re-engineering the DNA repair machinery for therapeutic purposes

Our work on repair of radiation-induced DNA damage has also led to an interest in how repair systems might be manipulated for therapeutic purposes. Robust hyperactivation of DNA repair pathways is a significant component of the oncogenic phenotype and is the underlying factor in resistance to radiation therapy. An ability to selectively inhibit DNA repair holds great promise for improving the response to clinical radiation therapy.

Our laboratory has investigated a novel approach using antibody fragments to inhibit DNA double-strand break repair in tumor cells. Antibody therapy is widely accepted in oncology, but is currently limited to extracellular targets.  We may have found a way to overcome this limitation by coupling an antibody fragment via a scissile, or “self-immolative,” disulfide bond to a ligand that engages a receptor on the surface of the tumor cell. The ligand-antibody complex enters the cell by receptor-mediated endocytosis, dissociates, and the antibody fragment then escapes and enters the nucleus to bind its target. We recently reported proof in concept that this approach works, using a folate-antibody complex to approximately double the sensitivity of cancer cells to radiation. There are no fundamental barriers to clinical translation of this approach; indeed folate-mediated delivery is already in clinical trials for other classes of anti-tumor agents.

With the move to Emory University, we hope to expand these studies by creating and testing antibodies against other components of the DNA double-strand break repair machinery, by testing other methods for intracellular delivery, and by investigating the pharmacokinetics, pharmacodynamics, and efficacy of ligand- antibody conjugates in preclinical animal models.

We are also exploring the possibility of using a similar protein delivery technology to transport engineered, sequence-specific nucleases into living cells. In this case, the objective is to create a targeted double strand break in or near a defective gene and engage the homologous repair machinery to correct the defect using a wild-type donor template. While the underlying concept is scientifically well established, the practical issues surrounding nuclease delivery have hindered clinical translation.  As part of a multi-institutional NIH-designated Nanomedicine Development Center, we have achieved remarkable success in creating ligand-nuclease constructs for intracellular delivery of zinc-finger and TAL effector nucleases  (ZFNs and TALENs).  The Center’s plan is to introduce these nuclease to human hematopoietic stem cells for correction of sickle cell anemia and other hematopoietic disorders. Our move to Emory University consolidates three of the Center investigators on the Emory/Children’s Hospital of Atlanta campuses and two others at the Georgia Institute of Technology, in the joint Wallace Coulter Department of Biomedical Engineering.

Other research activities

Over the last decade, the laboratory has been involved in several other areas of research, notably including a multi-year, multi-investigator project using two-dimensional gel electrophoresis to identify biomarkers for colorectal, cervical, and head and neck squamous cell cancers.  This work led to eight publications and two patent applications. It also afforded training opportunities for two PhD students, a postdoctoral fellow, and an otolaryngology resident who subsequently joined the GHSU faculty as an independent investigator.

The laboratory also carried out preliminary studies with a remarkable new ceramic material, consisting of porous-wall, hollow glass microspheres.  These were originally developed within the US Department of Energy research complex. Our work shows that the microspheres hold unique promise as a drug delivery vehicle. These studies, which were carried out jointly with the Savannah River National Laboratory, led to one peer-reviewed publication, several other publications, and a prestigious award from Research and Development magazine. We have worked with the Georgia Research Alliance and Georgia Health Sciences University to secure intellectual property and to market the technology.

An acknowledgment

None of the laboratory’s accomplishments would have been possible without the dedicated work and intellectual contributions of our more than 50 undergraduate, graduate, and postdoctoral trainees. In addition, as Executive Editor of Nucleic Acids Research for more than ten years, I have had many interactions with scientists in my field, some of which seeded the collaborative projects described here.