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 Top2? and Top2?. The two enzymes have distinct biological functions with Top2? having unique roles in transcription and chromosome structure. Top2? 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 Top2? 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 Top2? mutations in patients with autism spectrum disorders (ASDs). In preliminary data, we show that the Top2? 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 Top2? proteins in neuronal cells. Our collaborator Peter McKinnon, St. Jude Children's Hospital, has generated mouse ES cells that express two distinct Top2? 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 Top2? 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.
DNA topoisomerases are enzymes that play key roles in transcribing genes in neurons, and loss of function of these enzymes may result in abnormal neuronal development and function. Another aspect of topoisomerases is that their normal reaction has the potential of generating DNA damage, and DNA damage may also result in abnormal neuronal development and function. Since topoisomerase mutations have been found in patients with autism spectrum disorders, we are trying to distinguish whether these mutations cause their effects form loss of topoisomerase function or the ability of topoisomerases to damage DNA.
