Thursday, April 18, 2024
4/18/2024
DNA topoisomerase II (Top2) carries out changes in DNA structure needed for efficient transcription, replication, and DNA repair. This enzymes introduce transient double strand breaks in DNA through a protein/DNA covalent intermediate termed the cleavage complex. The DNA cleavage mechanism of Top2 allows cells to catalyze changes in DNA conformation without the dangers of frank DNA double strand breaks. Mammalian cells contain two Top2 isoforms termed Top2a and Top2ß. The two enzymes have distinct biological functions with Top2ß having unique roles in transcription and chromosome structure. Top2ß is particularly important for transcription of neuronal genes. Interestingly, topoisomerases have recently been suggested to be particularly important in the transcription of long genes, suggesting that topoisomerase function may be uniquely important in neuronal cells. While the Top2 catalytic mechanism typically avoids generating double strand breaks, in some contexts, it has been suggested that Top2ß makes long lasting double strand breaks during the process of regulated transcription initiation. Recent results have also suggested that long lasting topoisomerase covalent complexes may cause neurotoxic DNA damage. Two recent reports have identified identical heterozygous Top2ß mutations in patients with autism spectrum disorders (ASDs). In preliminary data, we show that the Top2ß mutation found in the two independent ASD patients generates spontaneous DNA damage through the generation of enzyme mediated DNA strand breaks. In this application, we propose to explore the biochemical characteristics of this type of enzyme using in vitro enzyme assays and expression of the mutant enzyme in yeast and mammalian cells. A second aim of our studies is to study DNA damaging Top2ß proteins in neuronal cells. Our collaborator Peter McKinnon, St. Jude Children's Hospital, has generated mouse ES cells that express two distinct Top2ß mutations that in vitro lead to elevated topoisomerase mediated cleavage. We plan to characterize DNA damage responses and developmental defects when these cells differentiate into neuronal lineages. Finally, we propose to initiate development of a model system using human neuroblastoma SH-SY5Y cells to introduce topoisomerase mutations capable of generating spontaneous DNA damage into human neuronal cells. We will determine whether DNA damaging mutations of Top2ß have similar effects to loss of function mutations, or whether the generation of topoisomerase induced DNA damage leads to unique effects on neuronal cell differentiation and survival. These studies will highlight potential roles of endogenous DNA damage in human neurological diseases, and provide a model system that can be used to explore unique ways that topoisomerase mis- functioning can contribute to human diseases.
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