Use of the Z-DNA binding domain of ADAR1 as a probe for nucleic acids in left-handed conformation

• Main Author: Li, Heng
• Other Authors: Peter Droge
• Format: Theses and Dissertations
• Language: English
• Published: 2009
• Subjects: DRNTU::Science::Biological sciences::Human anatomy and physiology::Deoxyribonucleic acids
• Online Access: https://hdl.handle.net/10356/15657
• Institution: Nanyang Technological University

Both DNA and RNA can be in Z-conformation. Compared to classical B-DNA conformation, Z-DNA is in a higher energy state and studied extensively, so far mainly in vitro. It has been recognized that Z-DNA plays roles in many biological activities, including regulation of transcription, chromatin remodeling, genetic instability, etc. Bioinformatics analyses and experimental studies on particular genomic loci suggest that the Z-DNA forming sequences are enriched in gene regulatory regions, where the formation of Z-DNA is drived by transcription-induced negative supercoiling. However, the global distribution of Z-DNA in the human genome in vivo is still experimentally elusive, due to instable nature of Z-DNA and lack of Z-DNA specific probe. Here, we show for the first time the distribution of Z-DNA hotspots in the genome of cultured human cancer cells A549 by exploiting the Zα domain from human ADAR1 (ZαADAR1, simplified as Zα for convenience in the thesis) as a Z-DNA specific probe with a novel experimental strategy. A protocol for in vitro chromatin affinity precipitation (ChAP) was developed and a Z-DNA library was constructed and sequenced. In total, 186 Z-DNA hotspots were identified. Enrichment of hotspots in gene regulation regions was not found. Unexpectedly, 46 hotspots localize to the centromeric regions. Further examination on hotspots therein suggests that Z-DNA hotspots are strongly correlated with high occurrences of SNPs (single nucleotide polymorphism). Bioinformatics analysis using a Z-DNA prediction program shows that most of these Z-DNA forming hotspots (143 out of 186) could be transited to Z-conformation only at σ < - 0.08, suggesting particular regions in the centromeres contain high negative superhelical torsional strain. Genetic instability and gene conversion promoted by Z-DNA provide a new clue for the rapid evolution of the centromere. Z-RNA shares many similar structural features with Z-DNA. But so far its existence in vivo and potential biological roles are still elusive. In the second part of this study, we found that Zα could bind to RNA in vivo which was identified as rRNA. Further studies suggested that Zα could bind to mammalian ribosomes in a conformation-dependent manner. The effect of the binding of Zα to ribosomes was examined and Zα could inhibit in vitro translation efficiently.