Role of folding kinetics of secondary structures in telomeric G-overhangs in the regulation of telomere maintenance in Saccharomyces cerevisiae

Katarina Jurikova,Martin Gajarsky,Mona Hajikazemi,Jozef Nosek,Katarina Prochazkova,Katrin Paeschke,Lukas Trantirek,Lubomir Tomaska

doi:10.1074/jbc.ra120.012914

Abstract

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.

Highlights

The ends of eukaryotic chromosomes typically contain a 39 ssDNA G-rich protrusion (G-overhang)
Telomere shortening is most often prevented via extension by telomerase, a ribonucleoprotein enzymatic complex that includes an RNA template and a reverse transcriptase subunit, followed by the fill-in synthesis of the C-rich strand mediated by primase, Pol a, which maintains the number of tandem repeats within a relatively stable interval [13,14,15,16]
That the shapes of the CD spectra of ONG1-6 and ONG910 were indicative of having parallel and antiparallel G4 structures, respectively, the detailed analysis of spectral data revealed that these constructs are similar to ONG7 and ONG8 and capable of adopting at least two distinct G4 topologies that co-exist as a mixture of dominant and minor G4 species

Summary

Introduction

The ends of eukaryotic chromosomes typically contain a 39 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 DNAbinding proteins, such as Cdc in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Telomere shortening is most often prevented via extension by telomerase, a ribonucleoprotein enzymatic complex that includes an RNA template and a reverse transcriptase subunit, followed by the fill-in synthesis of the C-rich strand mediated by primase, Pol a, which maintains the number of tandem repeats within a relatively stable interval [13,14,15,16]

Results

Discussion

Conclusion