G-quadruplex-R-loop interactions and the mechanism of anticancer G-quadruplex binders

Abstract

Genomic DNA and cellular RNAs can form a variety of non-B secondary structures, including G-quadruplex (G4) and R-loops. G4s are constituted by stacked guanine tetrads held together by Hoogsteen hydrogen bonds and can form at key regulatory sites of eukaryote genomes and transcripts, including gene promoters, untranslated exon regions and telomeres. R-loops are 3-stranded structures wherein the two strands of a DNA duplex are melted and one of them is annealed to an RNA. Specific G4 binders are intensively investigated to discover new effective anticancer drugs based on a common rationale, i.e.: the selective inhibition of oncogene expression or specific impairment of telomere maintenance. However, despite the high number of known G4 binders, such a selective molecular activity has not been fully established and several published data point to a different mode of action. We will review published data that address the close structural interplay between G4s and R-loops in vitro and in vivo, and how these interactions can have functional consequences in relation to G4 binder activity. We propose that R-loops can play a previously-underestimated role in G4 binder action, in relation to DNA damage induction, telomere maintenance, genome and epigenome instability and alterations of gene expression programs.

Figure 1.

(A) The structure of a guanine quartet with a stabilizing K+ in the center channel. Below, the canonical potential G4-forming sequence (PQS). G, guanine; N, any nucleotide. (B) Different G4 structural conformations: (I) intra-strand parallel; (II) intra-strand anti-parallel; (III) inter-strand anti-parallel; (IV) inter-strand parallel. (C) NMR structure of a PQS present in the human KRAS proto-oncogene promoter (RCSB PDB ID: 5IV) (176).

Figure 2.

Chemical structures of G4 binders.

Figure 3.

(A) Molecular model of the interplay among G4, R-loop, DNA supercoiling and protein factors. Top, main factors that contribute to prevent or promote R loop formation. Below, G4s and factors binding single-strand DNA can stabilize the displaced strand of R-loops. DNA and RNA are shown in green and red, respectively. (B) Genomic maps of G4-binder-induced R-loops and PQS established experimentally with a polymerase-stop assay (27) at the RRN3P3 gene locus. The graphs show normalized genomic R-loop profiles for control (blue line) and FG-treated (red line) U2OS cells (71) and oriented PQS (light blue boxes with white arrow).

Figure 4.

Mechanisms of DNA breakage formation induced by G4 stabilization. (A) G4 can be bound by a nuclease (DNA2) that then cleaves the DNA strand. (B) G4s can be a barrier to DNA synthesis by DNA polymerases leading to either fork collapse and DNA damage or re-priming by a second DNA polymerase downstream to the G4 generating a single-strand break (SSB). The SSB then becomes a double-strand break (DSB) at the next round of DNA replication in daughter cells. (C) G4s can stabilize co-transcriptional R-loops which can cause replication/transcription conflicts (top) or activate a repair pathway generating DNA breaks (below). DNA and RNA are shown in green and red, respectively.