DNA damage that blocks transcription can prevent the expression of essential genes, leading to mutations, apoptosis, or necrotic cell death. Transcription-coupled repair is a cellular process by which some forms of DNA damage are repaired more rapidly from transcribed strands of active genes than from nontranscribed strands or the overall genome. Cockayne syndrome patients are characterized by developmental and neurological deficiencies and are specifically defective in the process transcription-coupled repair. It has been widely speculated that the transcription-coupled repair of oxidative-DNA lesions, in particular, may be an underlying cause of the underlying developmental and neurological deficiencies in Cockayne's syndrome, and may be involved in other diseases that involve the progressive loss of neurological function, such as Parkinsons and Alzheimer's disease. However, the rapid kinetics of oxidative repair relative to transcription, and the apoptotic cascade induced by reactive oxygen and stalled transcription machinery have made it technically difficult to address this hypothesis in mammalian cells, despite intense efforts. We therefore, propose to test this hypothesis directly in the model organism of E.coli, where the process of transcription-coupled repair and oxidative DNA repair are highly conserved. We show that the low complexity genome, well-characterized transcriptional operons, and use of purified DNA glycosylases and isogenic mutants allow us to overcome the obstacles arising in human cell cultures to detect and definitively answer this important question. We hypothesize that specific oxidative DNA lesions are repaired in a transcription-coupled manner in vivo. We further hypothesize that lesions that block RNA polymerase will be subject to transcription-coupled repair, whereas nonblocking lesions will not, and that the process will depend on a number of gene products including, a coupling factor- Mfd, nucleotide excision repair, and specific DNA glycosylases. To test these hypotheses, we will 1) use purified DNA glycosylases with known substrate specificities to measure the repair kinetics of different oxidative DNA lesions in vivo;2) examine the repair rates of different classes of oxidative damage, 8-oxoguanine, thymine glycol, and others, to identify which classes of oxidative lesions are repaired in a transcription-coupled manner;3) measure the repair rate of oxidative lesions and recovery of RNA synthesis in isogenic mutants that lack nucleotide excision repair, oxidative DNA glycosylases, or Mfd.
The results from this project will enhance our understanding of the roles of transcription and transcription-coupled repair in processing oxidative DNA damage that have been implicated in human disease. Reactive oxygen species are directly or indirectly associated with a range of human hereditary diseases ranging from Parkinsons and Alzheimers, to amyotrophic lateral sclerosis and Friedreich's ataxia, to Fanconi anemia and Cockayne syndrome. In addition, there is increasing evidence to suggest reactive oxygen species play a significant role in the spontaneous cancers and aging. Since both oxidative DNA damage and transcription arrest generate strong signals for apoptosis, the research may lead to novel modes of chemotherapy, involving selective inhibition of transcription-coupled repair in target cells combined with administration of transcription-blocking drugs or antioxidants.
|Courcelle, Justin; Wendel, Brian M; Livingstone, Dena D et al. (2015) RecBCD is required to complete chromosomal replication: Implications for double-strand break frequencies and repair mechanisms. DNA Repair (Amst) 32:86-95|
|Jeiranian, H Arthur; Schalow, Brandy J; Courcelle, Charmain T et al. (2013) Fate of the replisome following arrest by UV-induced DNA damage in Escherichia coli. Proc Natl Acad Sci U S A 110:11421-6|
|Schalow, Brandy J; Courcelle, Charmain T; Courcelle, Justin (2012) Mfd is required for rapid recovery of transcription following UV-induced DNA damage but not oxidative DNA damage in Escherichia coli. J Bacteriol 194:2637-45|
|Schalow, Brandy J; Courcelle, Charmain T; Courcelle, Justin (2011) Escherichia coli Fpg glycosylase is nonrendundant and required for the rapid global repair of oxidized purine and pyrimidine damage in vivo. J Mol Biol 410:183-93|