Genome Instability in Yeast: Getting at the Mechanisms of Genetic Change
In my 20 years here at the University of Arizona, I now find myself in a fascinating as well as mysterious field of study, that of genome instability. Chromosomes, the source of information that dictates a cells behavior, are complex and fascinating structures. As we now know, information in chromosomes directs the synthesis of RNA and protein molecules that more directly affect cell behavior. Changes to chromosomes changes that protein and RNA-encoded information, and thus changes a cells behavior. Chromosome changes are key to both how organisms evolve (long time scale) and how pathology arises (short time scale). As a graduate student some 30 years ago, I first embarked on the study of chromosome change in bacteria (ref1). As a postdoc I discovered a key control called checkpoints that coordinates DNA repair and the cell cycle, and normally prevents chromosome changes (ref 2,3,4,5,6). My laboratory now is devoted to the study of chromosome change in eukaryotic cells.
To study chromosome change, we currently use the model organism S. cerevisiae, called budding yeast. We plan to extend our studies to other cell types as principles of instability become clear. Chromosome changes in all organisms are often associated with errors committed by the replication fork, a complex structure that duplicates DNA each cell division (Figure 1). When this machin, and/or associated other molecular machines, makes an error, large-scale chromosome changes arise wherein two chromosome segments are joined that were not previously adjacent (Figure 2). Large-scale changes are now known to be prevalent in many cancer cells (ref7). An early and famous example is the translocation called the Philadelphia Chromosome, resulting in altered gene function and leukemia.
We have developed systems in budding yeast that allow us to readily identify chromosome changes and study how they were formed. We do a lot of genetics, introducing genetic mutations in specific checkpoint and DNA replication proteins and then measuring chromosome change. Our studies have encompassed the areas of replication fork biology, DNA repair and genome structure. There is considerable literature in all three, and it takes considerable intellectual effort to make sense of how each contributes to genome instability.
Recently we have made two major findings that we continue to pursue. (9-12). First, we found that dicentrics, that is, chromosomes with 2 centromeres, play a major role in large-scale changes. Dicentrics have long been known to be associated with chromosome changes (ref13), yet how they are normally prevented, and how they form when prevention fails, is unknown and a very active area of research. Second, we are assimilating what is known about replication fork proteins with our studies of the roles of these proteins in preventing chromosome changes. We formulate and test specific models of how specific proteins normally prevent instability. We summarze our study of > 50 protein, acting in a dozen different protein complexes and systems, into a heat map that reveals the relative contributions of replication fork systems to genome stability (Fig3). We continue to develop and test the connections between replication fork systems and genome instability (14 review).