Stability of DNA G-quadruplexes in cell lysates

Publikation: KonferencebidragPosterForskning

DNA G-quadruplexes are non-canonical secondary structures that can form in guanine-rich sequences. Potential G-quadruplex forming sequences have been found in telomere and gene promoter regions of the human genome, and are potential anti-cancer drug targets (1). G-quadruplexes have been reported to interact with a number of proteins and to be unfolded by various helicases (2), however their observation and characterization in vivo is rather limited. In order to investigate the stability of G-quadruplexes in a complex protein environment, we simulate cellular conditions using HeLa cell lysates. We use Förster resonance energy transfer (FRET) spectroscopy to monitor the stability of several single stranded DNA substrates containing G-quadruplex motifs and gel electrophoresis to visualize DNA substrate integrity and eventual nuclease-induced cleavage. We observe that G-quadruplex unfolding and DNA cleavage are stronger in the presence of ATP and MgCl2. Our results suggest that G-quadruplex stability in cellular lysates is affected by nuclease cleavage and G4 binding/unfolding proteins. We have previously observed that G-quadruplexes undergo transient folding in buffer solutions with low ionic strength (3). Using single molecule FRET, we plan to investigate G-quadruplex folding/unfolding dynamics in cellular extracts. (1) Rhodes, D., & Lipps, H. J. (2015). G-quadruplexes and their regulatory roles in biology. Nucleic acids research, 43(18), 8627-8637.(2) Brázda, V., Hároníková, L., Liao, J. C., & Fojta, M. (2014). DNA and RNA quadruplex-binding proteins. International journal of molecular sciences, 15(10), 17493-17517.(3) Aznauryan, M.; Sondergaard, S.; Noer, S. L.; Schiott, B.; Birkedal, V., (2016). A direct view of the complex multi-pathway folding of telomeric G-quadruplexes. Nucleic acids research, 44, 11024-11032.
Udgivelsesår28 aug. 2018
StatusUdgivet - 28 aug. 2018
Begivenhed2018 Cold Spring Harbor meeting: Single Biomolecules - Cold Spring Harbor Laboratory, Cold Spring Harbor, USA
Varighed: 28 aug. 20181 sep. 2018


Konference2018 Cold Spring Harbor meeting: Single Biomolecules
LokationCold Spring Harbor Laboratory
ByCold Spring Harbor

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