Project Summary The focus of my lab is to understand the mechanisms of genome instability caused by structure-prone DNA repeats. We are particularly interested in the mechanisms of repeat expansions that are responsible for over fifty hereditary diseases in humans. Recent advances in long-read sequencing revealed a new paradigm: massive genome-wide expansions of structure-prone DNA repeats in human cancers. Thus, understanding the mechanisms responsible for large-scale repeat expansions is fundamentally important and has broad biomedical implications. My lab was the first to show that expandable DNA repeats stall replication fork progression in every experimental system studied, including bacteria, yeast, and human cells. This led us to propose that repeats can be added while replication fork escapes from a “repetitive trap”. We first confirmed this idea in a yeast experimental system. Depending on the mode of replication fork progression through a repeat, expansions occur by incorporation of unprocessed flaps during Okazaki fragments maturation, replicative or post-replicative template-switching, or break-induced replication. Recently, we developed a new experimental system to study large-scale expansions in human cells, which implicates DNA replication as well. Repeat expansions also occur in terminally differentiated somatic cells that do not undergo DNA replication. We have, therefore, adjusted our experimental system to study repeat expansions in non- dividing yeast cells. Our results point to DNA nick repair as a possible mechanism. We plan to move our research in several directions. First, we will elucidate the role of DNA nick repair in expansions of Friedreich’s ataxia (FRDA) (GAA)n repeats in dividing and non-dividing yeast cells by introducing targeted nicks with Cas9 nickases. We will establish its genetic controls and study the role of the human FAN1 nuclease expressed in yeast. Second, we will unravel the mechanisms of large-scale repeat expansions in human cells by conducting candidate gene analysis in a plasmid system utilizing SV40 replication machinery. We will also study the effects of compounds that disrupt or stabilize DNA triplexes formed by these repeats on their replication and expansion. We will further extend our studies into a different system based on the EBNA1-dependent replication which involves regular cellular replication fork. Third, we will carry out structure-functional analysis of other expandable homopurine-homopyrimidine repeats, including the (AAGGG)n repeat, which is responsible for cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS), the (CCCTCT)n repeat that modifies the expressivity of X-linked dystonia parkinsonism (XDP), and the (GAAA)n repeat, which recurrently expands in many human cancers. At present, there are no data on DNA structures formed by those repeats or on the mechanisms of their expansions. We will address these matters by using the broad arsenal of methods and experimen...