Sunday, May 5, 2024
5/5/2024
Project Summary/Abstract Approximately half the human genome is comprised of repetitive DNA sequences that are thought to control a wide range of cellular functions. DNA repeats are found throughout the genome, and are polymorphic in length due to their genetic instability. Mutation rates of repeat elements are 101-105 fold higher than in other parts of the genome, and is triggered by the formation of transient unusual DNA structures (extrahelical extrusions) during DNA metabolic processes. The detrimental consequences of repeat instability are exemplified by triplet repeat expansions that cause a number of neurodegenerative diseases such as Huntington’s disease, Friedreich’s ataxia and Fragile X related disorders. The rate of expansion of triplet repeats is proportional to repeat length and sequence homogeneity. DNA repair mechanisms have evolved to maintain genomic stability, and protect the DNA from damage caused by environmental agents. One such process is DNA mismatch repair (MMR), a highly conserved antimutagenic pathway that maintains the stability of the human genome by correcting replication errors and preventing chromosomal rearrangements. Unexpectedly, a mutagenic non- canonical function of MMR has been implicated as the cause of triplet repeat expansions. Loss of MMR function attenuates triplet repeat expansion, although the molecular mechanisms of this non-canonical MMR activity are poorly understood. However, this mutagenic action of MMR requires the proteins MutSb and MutLa (and possibly MutLg). FAN1 is a deoxyribonuclease that was originally identified as a factor involved in the repair of DNA interstrand crosslinks. Loss of FAN1 function exacerbates repeat expansion, suggesting a role for FAN1 in suppression of triplet repeat expansion by mechanisms that are not understood. We are interested in the molecular mechanisms responsible for the crosstalk between these opposing effects of MMR and FAN1 in the control of mutation production within triplet repeats. We have discovered a novel activator of the FAN1 nuclease on triplet repeat extrusions, a finding that represents the first step not only in our understanding of the mechanism of FAN1 action, but also in our quest to develop a unified understanding of the mechanism of triplet repeat expansion by integrating biochemical, cellular, and genetic studies. In Aim 1, we will elucidate the molecular features of FAN1 nuclease function by evaluating the modulatory effects of DNA sequence/structure and protein co-factors. In Aim 2, we will dissect the role of protein-DNA and protein-protein interactions in the repair of triplet repeat extrusions by FAN1. In Aim 3, we will use unbiased proteomic approaches to identify co-factors that facilitate FAN1 nuclease function in a cellular milieu. Completion of these studies will shed light on the pathways that modulate triplet repeat expansion, and will have implications more broadly for the mechanisms of genome instability.
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