Sequence Specificity of Transient Hoogsteen Base-Pairs in Canonical Duplex DNA and Z-DNA Formation.

[摘要] Sequence-specific DNA flexibility provides an additional layer of the genetic code. Using Nuclear Magnetic Resonance R1ρ relaxation dispersion we recently discovered that Watson-Crick (WC) base-pairs in flexible CA/TG and TA/TA steps of DNA transiently morph into Hoogsteen (HG) base-pairs in the context of canonical duplex DNA. CA/TG and TA/TA steps are the most flexible of all 10 dinucleotide steps. We designed DNA duplexes to contain flexible and rigid dinucleotide steps and strikingly obtained evidence for transient Hoogsteen base-pairs in all contexts examined. We observe small, significant variations in the population and lifetime of transient HG base-pairs that are anti-correlated to WC dinucleotide step stabilities. Phi-value analysis suggests a late transition state possibly involving a syn purine base. The broad and robust occurrence of transient HG base-pairs suggests their broad presence in the genome. In contrast to the sequence specificity of HG base-pairs, Z-DNA formation is favored in rigid and stable dinucleotide steps, specifically purine-pyrimidine repeats. Interestingly, transient, lowly populated HG base-pairs can still be observed in a Z-DNA forming sequence despite the increased dinucleotide step stability. We monitored the salt- and human adenosine deaminase- (hADAR1-Zα) induced B-to-Z-DNA transitions in a CpG hexamer using Circular Dichroism (CD) to monitor global DNA conformational changes. Time-resolved CD measurements revealed a biphasic B-to-Z-DNA transition consisting of fast and slow phases for both salt- and Zα-induced transitions. These data are consistent with a B-to-Z-DNA transition involving at least two sequential steps. Uniform 5-Methylcytosine modification increases the fast phase two-fold while N1-methylguanine and 7-deazaguanine modifications cause unique structural perturbations precluding B-Z transient kinetics that potentially reveal new features of HG dependent DNA flexibility and structure. Overall, the findings presented in this thesis reveal an additional widespread layer to the genetic code and call for a deeper understanding of their behavior when adjacent to one another for further insight into potential roles in vivo.

[发布机构] University of Michigan

[效力级别] Watson-Crick and Hoogsteen Base Pairs

[关键词] Nucleic Acid Dynamics;Watson-Crick and Hoogsteen Base Pairs;Alternative DNA Conformations;B-DNA to Z-DNA Transition Using Circular Dichroism;Nuclear Magnetic Resonance;Kinetics;Chemistry;Science;Chemistry