Evidence for G-quadruplex-based telomere capping and the presence of G-quadruplex DNA genome-wide in Saccharomyces cerevisiae.

摘要

G-quadruplex DNA, or G4 DNA, is non-canonically base paired DNA that is composed of stacked planar tetrads of Hoogsteen hydrogen-bonded guanines. Evidence for these alternately based paired guanine structures has existed for half a century, yet whether G4 DNA plays positive, functional roles in natural biology remains an important and open question today due to the paucity of direct evidence. Perhaps the best characterized location at which these structures may have biological relevance is at telomeres, the nucleoprotein caps of linear chromosomes. The G-quadruplex forming potential (QFP) of telomeres is highly conserved, from yeast to humans, and G-quadruplexes have been identified in vivo in the protist Stylonychia lemnae. However, a clear functional role for telomeric G4 DNA has not been characterized yet, and was part of the goal of this thesis work. To assess the functional relevance of these structures at eukaryotic telomeres, we employed a budding yeast mutant, cdc13-1, that is defective for telomere capping by the protein Cdc13. We show that a variety of mechanisms that stabilize G4 DNA are able to reconstitute functional telomere capping in this system. The role of Cdc13 itself in telomere capping was assessed in further detail using novel mutations to the N-terminal DNA binding and dimerization domains. In addition, we have worked on designing methods to assess the role of G4 DNA throughout the genome including affinity chromatography using the highly specific G4 DNA ligand, N-methylmesoporphyrin IX (NMM) and genome-wide ChIP-on-chip tiling array analysis of G4 DNA binding proteins. With the latter methodology, we have found that G4 DNA proteins associate with normal telomeres, ribosomal DNA (rDNA) and various other regions of the yeast genome possessing QFP, which suggests that G4 DNA may have functional roles throughout the genome in a normal biological setting, and warrants future investigation.