Overview of Research Area
Cancer is the end result of genetic mutations that lead to a growth advantage and expansion of rare cellular clones. Genetic mutations, in turn, represent the failure of a cell to correctly repair the DNA lesions that are caused by a variety of damaging agents. Specifically, inefficient repair of DNA double-strand breaks (DSBs) leads to a persistence of lesions that ultimately become substrates for chromosomal rearrangement, a hallmark of malignancy (Figure 1). The long-term goal of our laboratory is to evaluate the hypothesis that different types of DSB repair deficiencies lead to the accumulation of distinct DNA lesions or repair intermediates, and that these have a differential likelihood of contributing to rearrangement. This will require a detailed understanding of the nature of DSB intermediates and repair processes.
Figure 1. A color-coded spectral karyotype (SKY) image of chromosomes from a mouse cancer (courtesy of Dr. David Ferguson). Rearrangements typical of cancer are evident as bi- and tri-colored chromosomes.
To achieve our goal, we are systematically examining both enzymatic and structural DSB repair proteins and how these interact to achieve the sequence of biochemical events that results in repair (Figure 2). This is being done using novel assays developed in the genetically tractable model organism Saccharomyces cerevisiae, since it is now clear that the fundamental mechanisms of DSB repair and rearrangement are preserved in all eukaryotes. Areas of particular interest are first to examine the factors that impact the choice between the two major pathways of DSB repair (homologous and nonhomologous). Second, we are continuing to identify and characterize the enzymes that process damaged terminal bases and ligate DSB ends. Third, we are working to establish how these various proteins interact to create the nonhomologous end-joining (NHEJ) "repairosome". Finally, we are examining how perturbations in the above impact the development of chromosomal aberrations.
Figure 2. The pathways of DSB repair. NHEJ, our favorite pathway, entails the direct rejoining of ends. Recombination uses a donor template to accurately repair the break.
Some specific areas of current active interest
In a collaborative project with Dr. Tom Glover which was recently selected by the NIEHS for Challenge grant funding, we are using next generation sequencing and microarray technologies to explore the mechanisms by which replication inhibition and other genotoxic stresses leads to chromosomal rearrangement. Such mutations are intimately related to the phenomenon of chromosome fragile sites which Dr. Glover has studied for many years, as well as to the DNA repair mechanisms targeted by the Wilson laboratory. High throughput sequencing, now routinely available on campus, provides an outstanding means of exploring the contributions of different DSB repair pathways to chromosomal rearrangement. We have developed both wet lab tools as well as a novel software platform for this approach:
and will exploit these in the coming years to understand the mechanistic underpinnings of chromosome rearrangements.
Assembly of the NHEJ repair complex at a DSB
There are 8 yeast proteins known to be required for all NHEJ repair events, and some others that are brought in as needed. Figure 3 shows a reasonable structural representation of these proteins. It is clear that there is a lot of protein architecture there for not so much DNA. How do all of these proteins interact to achieve repair? Do they all bind at once, or is the reaction ordered in some way? How? We are addressing these questions by performing chromatin immunoprecipitation of protein binding to DSBs (and related techniques) and discerning the exact nature of the interactions between the NHEJ proteins.
Figure 3. The proteins that catalyze yeast NHEJ (top) and structural representation of closely homologous proteins (bottom). The problem is very much like a puzzle – how to put all of these pieces together in a productive fashion?
Basis of the unique enzymology of NHEJ enzymes
The NHEJ reaction seems deceptively simple as it just rejoins existing DSB ends. However, from a catalytic standpoint, polymerase and ligases acting at DSBs do something fairly remarkable and unique to NHEJ – they perform reactions on two DNA molecules that are, by definition, not supported by an intact template strand (illustrated for polymerases in Figure 4). Thus, the enzyme itself must somehow provide the stability and binding energy to bridge the two DNA substrate molecules. This is both a fascinating problem in protein chemistry and a mechanism which is central to the disposition of DSBS among several possible pathways of DSB repair.
Figure 4. Summary of the synthetic reactions catalyzed by the NHEJ polymerase Pol4. Somehow the enzyme manages to fill the gap in a DSB even though the two sides of the DSB are NOT stably associate (as these drawing might incorrectly suggest).
Mechanisms of 5’ resection
When cells can’t complete NHEJ, they initiate homologous recombination. The first step of recombination is the degrade, or resect, the 5’ end of the DSB. This step is not only required for recombination, but simultaneously blocks any further possibility for accurate NHEJ. It is thus not surprising that resection is the key regulated step in the control of DSB repair pathway choice. Despite this, and years of research, the mechanism(s) of 5’ resection are poorly understood. The Mre11-Rad50 complex is clearly involved, but the multiplicity of actions of this complex have made this key regulated reaction difficult to describe. We have developed novel approaches for examining this reaction at high resolution in order to elucidate its mechanism.