Replication through DNA Structures and Consequences for Genome Stability

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Project Summary/Abstract Almost half of the human genome is composed of repetitive DNA elements, and about three percent of the genome is composed of microsatellites, short tandem repeats of 1-6 DNA bases. Many repeat sequences can form alternative DNA structures that interfere with replication and repair. This can lead to disease-causing repeat expansions, such as the CAG/CTG expansions that cause Huntington’s disease, myotonic dystrophy, and many spinocerebellar ataxias. Breaks within structure-forming repeats cause chromosome deletions and rearrangements, which are common in cancer cells undergoing replication stress. The goal of my laboratory is to study mechanisms of genome instability caused by structure-forming repeats, and to elucidate cellular pathways that have evolved to prevent these deleterious mutations. We recently discovered that long tracts of structure-forming CAG repeats relocate to the nuclear periphery to facilitate replication through the tract and prevent chromosome breakage and repeat expansions. This pathway depends on modification of proteins at the replication fork by sumoylation, and subsequent interaction of the sumoylated proteins with components of the nuclear pore complex (NPC) in late S phase, followed by release back into the nuclear interior in G2 phase. Recent data shows that this pathway is relevant for several types of replication barriers, including protein blocks and other structure-forming repeats. Therefore, it is vital to better understand the purpose of this relocation to the NPC and its role in facilitating replication and preventing genome instability, which is our long- term goal. We have developed a system to follow the location of an expanded CAG tract or other structure- forming repeats in the cell nucleus using microscopy, complemented by biochemical techniques to detect proteins interacting with the repeat locus. We will use the budding yeast (S. cerevisiae) system which allows us to combine these approaches with the powerful genetics of the yeast system. Yeast replication and repair pathways retain a high level of conservation with human cells, but the smaller size of the yeast genome and proteome and wild-type (non-transformed) state of cells are advantages that will allow us to make significant progress on our goals. We plan to elucidate the purpose of relocation of stalled replication forks to the nuclear pore complex and the mechanisms that occur there to allow restart of replication forks, using both established and novel approaches. We will also determine how nuclear pore-linked fork restart prevents chromosome breaks and repeat instability and determine how repeat expansions occur during fork recovery. Our aim is to understand NPC-dependent modification of replisome-associated proteins and fork remodeling that occurs at replication barriers, and in so doing understand vital cellular processes that maintain genome stability. This is important because understanding how mutations arise is critical to developing strategies to prevent their occurrence.