The Functional Consequences of Eukaryotic Topoisomerase 1 Interaction with G-Quadruplex DNA

Abstract

Topoisomerase I in eukaryotic cells is an important regulator of DNA topology. Its catalytic function is to remove positive or negative superhelical tension by binding to duplex DNA, creating a reversible single-strand break, and finally religating the broken strand. Proper maintenance of DNA topological homeostasis, in turn, is critically important in the regulation of replication, transcription, DNA repair, and other processes of DNA metabolism. One of the cellular processes regulated by the DNA topology and thus by Topoisomerase I is the formation of non-canonical DNA structures. Non-canonical or non-B DNA structures, including the four-stranded G-quadruplex or G4 DNA, are potentially pathological in that they interfere with replication or transcription, forming hotspots of genome instability. In this review, we first describe the role of Topoisomerase I in reducing the formation of non-canonical nucleic acid structures in the genome. We further discuss the interesting recent discovery that Top1 and Top1 mutants bind to G4 DNA structures in vivo and in vitro and speculate on the possible consequences of these interactions.

Keywords: G-quadruplex; genome instability; topoisomerase I.

Figure 1

Yeast WT Top1 and Top1Y727F bind to G4 structures. Western blots of pulldowns of WT Top1-3XFLAG (top) and Top1Y727F-3XFLAG (bottom) from yeast whole cell lysates with biotinylated DNA oligonucleotides (MilliporeSigma). Biotinylated oligonucleotides G4-1, G4-2, C, and T were conjugated to Streptavidin-Coupled M-280 Dynabeads. Following the mechanical lysis of yeast cells with Biospec Mini-bead-beater, the cell lysate was collected and sonicated. Oligo-conjugated Dynabeads were incubated at 4 °C overnight with the yeast extract, washed, and then eluted by boiling in 1XSDS-PAGE loading buffer followed by immunoblotting analysis using anti-FLAG antibody to detect 3XFlag-tagged Top1 or Top1Y727F.

Figure 2

A model of genome instability induced by co-transcriptionally formed G4 DNA and the effect of Top1 activity and mutation. RNAP—RNA polymerase complex. Dotted line—the nascent transcript. (−)—negative tension behind the transcription complex. (+)—positive tension ahead of the transcription complex. Top1mut—Top1 mutant.