Chromosome Stability Group
Many environmental factors put human health at risk through genomic destabilizing processes. The Chromosome Stability Group (CSG) located in the Laboratory of Molecular Genetics focuses on mechanisms that maintain or change chromosome stability.
All organisms possess intricate networks for genome duplication and maintenance that assure stability in the face of internal and external environmental stresses or during normal growth and development. The stability is provided through gene expression networks and protein interactions that assure coordination of normal replicative processes and responses to potential genome threats.
The CSG has created model systems in the budding yeast Saccharomyces cerevisiae and in human cells to address genome stability, the impact of the environment and underlying mechanisms. CSG investigates sources of spontaneous and damage-induced instability, molecular events in repair and resulting genomic changes.
Recombination, repair, and replication (the 3-R's) are intimately associated with many types of mutations and chromosome rearrangements. CSG investigations of the 3-R's in yeast have led to the development of several new approaches and concepts including direct RNA repair of DNA double-strand breaks (DSBs), ability to measure resection at random DSBs, mechanisms and consequences of altered lagging strand replication, as well as hypermutability by environmental agents at DSB resected ends.
The CSG in recent years has extended its previous substantial efforts on human genes and function in yeast-based systems to the development of an expanded capability to address factors affecting genome stability in human cells in vitro and ex vivo, with a focus on the p53 master regulatory network. The CSG recent studies have revealed a greatly expanded p53 universe of targeted genes, including many of the innate immunity Toll-like Receptor (TLR) genes, and it has developed new approaches to finding and functionally identifying noncanonical target sequences. Importantly, the CSG is addressing the functionality of cancer-associated p53 mutations and uncovering interactions with other master regulatory networks, a notable example being the integration of the p53, angiogenesis and estrogen receptor pathways.
Using a variety of recently developed genetic and physical approaches, along with the several CSG developments over the past decade, CSG continues to explore new relationships between DSBs, repair, signaling, cell progression and stress networks. These studies have resulted in novel mechanistic approaches to clinically relevant diseases that may have strong environmental components. Ultimately, the combined yeast and human cell approaches will influence the group’s understanding of environmental factors that put human genome stability and health at risk.
Double-Strand Breaks (DSBs), DNA Repair, Replication-Associated Changes, and Localized Hypermutability
DSBs induction, repair and genomic changes:
DSBs are major sources — both good and bad — of genetic change in humans, and can result from environmental agents as well as programmed events. Utilizing budding yeast, a good model for many human DSB activities, the CSG has found that DSBs can arise through processed events between closely-spaced single strand lesions and identified a coordinated mechanism for successful repair. While DSBs that are induced directly can be efficiently repaired, the CSG established that the vast majority of gross chromosomal arrangements produced by ionizing radiation in yeast are due to DSBs in repeated DNAs that can "reshape the genome."
To understand the importance of levels of repair components across the cell cycle, the CSG has developed an approach using tetraploid yeast strains in which the levels of essential genes, particularly the sister chromatid cohesin genes, can be varied over four-fold. The group found that cohesin is limiting in repair and that cohesin may control damage-induced loss-of-heterozygosity (LOH), an important genetic change in human cells and cancer. The CSG also pioneered an approach for addressing single molecule appearance of chromosome breaks. Current work has established that a combination of end processing proteins is essential in preventing transition from a DSB to a chromosome break.
A major challenge to the repair field is characterization of events at randomly induced lesions. To this end, a novel system based on breakage of a circular chromosome allowed investigation of one of the earliest steps in processing and repair of random DSBs, that of resection to generate single strand tails. A similar system is being developed in human cells to address directly DSBs, single strand breaks, repair and inhibitors. The system is expected to provide useful insights about the impact of environmental agents on DNA stability in human cells. Overall, the findings are important to human exposures, repair capabilities and therapies.
Replication of DNA is highly coordinated and accurate. The CSG has identified many DNA motifs (e.g., inverted repeats), environmental factors (e.g., cadmium) and genetic defects that put replicating DNA at risk for mutation and breakage. Recent efforts address lagging strand replication where millions of Okazaki fragments must be joined to prevent subsequent DSBs. The CSG established that the exceptional accuracy requires coupled activities of two nucleases centered around DNA Pol δ strand-displacement synthesis. Thus, endogenous and environmental factors that influence lagging strand maturation are likely to be genetically risky. On the flip side, they may have therapeutic value.
Damage-induced localized hypermutability:
Recently, the CSG established that common environmental agents such as UV and alkylating chemicals can increase mutation frequencies >1000-fold in short single strand (10-20 kb) regions of the yeast genome, leading to clusters of widely spaced mutations. The hypermutability is due to unrepairable damage in single-strand DNA regions that can arise at processed ends of DSBs, uncapped telomeres or uncoupled replication forks. Furthermore, the synergistic effect on mutagenesis between single-strand DNA and UV-induced lesions suggests that weak mutagens may be potentiated in single strand DNA. Importantly, the group found clusters of coordinated multiple mutations spread over 100 kb in chromosomes of yeast grown in the presence of small amounts of the alkylating agent, MMS. Thus, long ssDNA is extremely risky to genome stability. Hypermutable regions and mutation clusters are a likely source of genomic alteration, leading to disease, carcinogenesis and evolutionary changes.
The p53 Master Regulatory Network: Mutations, Variation and Evolution
The p53 tumor suppressor is a major regulator of DNA repair and damage checkpoints in humans. Importantly, most cancers are altered for p53 function. "Rheostatable" p53 expression systems have been developed in yeast and human cells to understand the functionality of p53 target sequences, wild type and mutant p53s, as well as the breadth of the p53 master regulatory network. The CSG has discovered a greatly expanded universe of p53 targets and human diversity as well as variations in responses to specific stresses such as UV and anticancer agents. The CSG approaches have provided functional analysis of cancer-associated functional mutants from a variety of tumors.
Furthermore, the CSG discovered that p53 can cooperate in cis with estrogen receptor to enhance responsiveness of normal and cancer-mutant p53s. Related to this, a single promoter SNP in the FLT1 gene was identified that brings together the angiogenesis, p53 and estrogen receptor pathways. The development of "rules" for what constitutes p53 target sequences, particularly a category of noncanonical targets, has opened new ways of thinking about the p53 tumor suppressor. Surprisingly, groups of genes have acquired functional p53 responsiveness late in evolution, including a set of DNA metabolic genes and many of the innate immunity-related Toll-like receptors (TLRs). Very recently these findings were transitioned to investigations with human cells ex vivo using blood samples from the newly developed NIEHS clinical research unit (CRU). These findings are helping to decipher the many roles that p53 plays in preventing genomic changes, reducing environmental stresses. The approaches are helping to identify agents that influence components of the network.