| Autor: |
Fleming AM; Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850 United States., Jenkins BLGC; Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850 United States., Buck BA; Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850 United States., Burrows CJ; Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850 United States. |
| Abstrakt: |
Molecular details for DNA damage impact on the folding of potential G-quadruplex sequences (PQS) to non-canonical DNA structures that are involved in gene regulation are poorly understood. Here, the effects of DNA base damage and strand breaks on PQS folding kinetics were studied in the context of the VEGF promoter sequence embedded between two DNA duplex anchors, referred to as a duplex-G-quadruplex-duplex (DGD) motif. This DGD scaffold imposes constraints on the PQS folding process that more closely mimic those found in genomic DNA. Folding kinetics were monitored by circular dichroism (CD) to find folding half-lives ranging from 2 s to 12 min depending on the DNA damage type and sequence position. The presence of Mg 2+ ions and the G-quadruplex (G4)-binding protein APE1 facilitated the folding reactions. A strand break placing all four G runs required for G4 formation on one side of the break accelerated the folding rate by >150-fold compared to the undamaged sequence. Combined 1D 1 H-NMR and CD analyses confirmed that isothermal folding of the VEGF -DGD constructs yielded spectral signatures that suggest formation of G4 motifs, and demonstrated a folding dependency with the nature and location of DNA damage. Importantly, the PQS folding half-lives measured are relevant to replication, transcription, and DNA repair time frames. |
