The ends of eukaryotic chromosomes typically contain a 3' ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing-based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.

Central European Institute of Technology Masaryk University Brno Czech Republic

Central European Institute of Technology Masaryk University Brno Czech Republic; Institute of Biophysics Czech Academy of Sciences Brno Czech Republic

Department of Biochemistry Faculty of Natural Sciences Comenius University in Bratislava Bratislava Slovakia

Department of Genetics Faculty of Natural Sciences Comenius University in Bratislava Bratislava Slovakia

Department of Oncology Hematology and Rheumatology University Hospital Bonn Bonn Germany

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Extraordinary diversity of telomeres, telomerase RNAs and their template regions in Saccharomycetaceae

Insight into formation propensity of pseudocircular DNA G-hairpins