DNA Topoisomerase Misfunctioning in Neurological Disease

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 Top2α and Top2β. The two enzymes have distinct biological functions with Top2β having unique roles in transcription and chromosome structure. Top2β 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 Top2β 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 Top2β mutations in patients with autism spectrum

disorders (ASDs). In preliminary data, we show that the Top2β 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 Top2β proteins in neuronal cells. Our collaborator Peter McKinnon, St. Jude Children's Hospital, has generated mouse ES cells that express two distinct Top2β 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 Top2β 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.