The Interplay of Replication and Transcription in Fragility of Structured DNA

Tufts University, Medford MA

Investigators

Abstract

The integrity of DNA is fundamental to life, but sometimes DNA can break, leading to loss of genetic material or even cell death. Interestingly, certain areas of chromosomes, known as fragile sites, tend to break more frequently. These fragile sites are a frequent source of DNA mutations, for example loss or gain of genetic material. Thus, an understanding of the basis of chromosome fragility will provide insight into causes of disease and of genome evolution. This topic is of great interest to college students studying molecular biology and genetics, and a Molecular Genetics Project Laboratory was developed to provide a research experience to 16 students a year, investigating the causes of chromosome fragility using a yeast model system. The goal of the course is to identify new pathways that protect against fragility, and give the students an authentic research experience. This project will support both the laboratory class as well as follow-up experiments by undergraduate and graduate students, to gain new knowledge about important pathways that prevent deleterious mutations. Many fragile sites in the human genome are associated with both DNA structures that interfere with replication and transcribed regions. It is suspected that the co-incidence of structure-forming DNA sequences, transcription, and replication may combine to cause these regions to be prone to fragility, however the interplay between them is poorly understood. Genetic systems have been developed in the yeast Saccharomyces cerevisiae to measure chromosome breaks that are induced at specific structure-forming repetitive DNA sequences that cause DNA fragility. In this system, transcription state and replication timing through the DNA structure can be controlled, to directly measure the consequences for DNA breakage. The role of proteins in causing or protecting against DNA breaks will be determined, including the action of identified nucleases Mlh1-Mlh3 and Mus81. These tools will be employed to elucidate the contributions and interplay of transcription and replication in causing fragility at two physiologically relevant fragile sequences: (1) expanded CAG repeats and (2) a long AT repeat from the human fragile site FRA16D. The knowledge gained will enhance understanding of mechanisms of fragility and the resulting genome instability that leads to chromosomal deletions and rearrangements. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.