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[Preprint]. 2024 Jan 21:2024.01.20.576387.
doi: 10.1101/2024.01.20.576387.

DNA Damage Accelerates G-Quadruplex Folding in a Duplex-G-Quadruplex-Duplex Context

Aaron M Fleming  1 , Brandon Leonel Guerra Castañaza Jenkins  1 , Bethany A Buck  1 , Cynthia J Burrows  1
Affiliations

DNA Damage Accelerates G-Quadruplex Folding in a Duplex-G-Quadruplex-Duplex Context

Aaron M Fleming et al. bioRxiv. .

Update in

Abstract

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 Mg2+ 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 1H-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.

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Figures

Figure 1.
Figure 1.
(A) Short-patch BER pathway illustrating the DNA damage structures studied within the VEGF-DGD. (B) The DGD construct along with (C) the modified VEGF inserts studied in this work.
Figure 2.
Figure 2.
Thermodynamically folded VEGF-DGD evaluated by CD (A) and 1D 1H-NMR (B). Monitoring of PQS isothermal folding in the DGD context in Li+ when 100 mM K+ was added by following the CD (C and D) and 1D 1H-NMR (E) spectral changes.
Figure 3.
Figure 3.
Folding half-lives for the VEGF-DGD construct with a central DNA damage when incubated in Li+ at 30 °C followed by addition of 100 mM K+ ions.
Figure 4.
Figure 4.
1D 1H-NMR and CD characterization of the VEGF-DGD motifs containing the DNA damage types (A) OG, (B) F, (C) 5’-pF, or (D) p(Gap).
Figure 5.
Figure 5.
Position-dependent folding half-lives for DNA-damage placed in either 5’-G or 3’-G runs of the VEGF-DGD motif.
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