Topoisomerase I plays a critical role in suppressing genome instability at a highly transcribed G-quadruplex-forming sequence.

Puja Yadav,Victoria Harcy,Nayun Kim,Sue Jinks-Robertson,Juan Lucas Argueso,Margaret Dominska,Philip C Hanawalt

doi:10.1371/journal.pgen.1004839

Abstract

G-quadruplex or G4 DNA is a non-B secondary DNA structure that comprises a stacked array of guanine-quartets. Cellular processes such as transcription and replication can be hindered by unresolved DNA secondary structures potentially endangering genome maintenance. As G4-forming sequences are highly frequent throughout eukaryotic genomes, it is important to define what factors contribute to a G4 motif becoming a hotspot of genome instability. Using a genetic assay in Saccharomyces cerevisiae, we previously demonstrated that a potential G4-forming sequence derived from a guanine-run containing immunoglobulin switch Mu (Sμ) region becomes highly unstable when actively transcribed. Here we describe assays designed to survey spontaneous genome rearrangements initiated at the Sμ sequence in the context of large genomic areas. We demonstrate that, in the absence of Top1, a G4 DNA-forming sequence becomes a strong hotspot of gross chromosomal rearrangements and loss of heterozygosity associated with mitotic recombination within the ∼20 kb or ∼100 kb regions of yeast chromosome V or III, respectively. Transcription confers a critical strand bias since genome rearrangements at the G4-forming Sμ are elevated only when the guanine-runs are located on the non-transcribed strand. The direction of replication and transcription, when in a head-on orientation, further contribute to the elevated genome instability at a potential G4 DNA-forming sequence. The implications of our identification of Top1 as a critical factor in suppression of instability associated with potential G4 DNA-forming sequences are discussed.

Highlights

In addition to the canonical Watson-Crick helical duplex (BDNA), genomic DNA, especially repetitive sequences, can assume other types of structures such hairpins, Z-DNA, triplex DNA (HDNA) or tetrahelical DNA structures [1,2,3,4,5,6]
We used a reporter system to quantitatively measure the level of genome instability occurring at a G4 DNA motif integrated into the yeast genome
We showed that the disruption of Topoisomerase I function significantly elevated various types of genome instability at the highly transcribed G4 motif generating loss of heterozygosity and copy number alterations, both of which are frequently observed in cancer genomes

Summary

Introduction

In addition to the canonical Watson-Crick helical duplex (BDNA), genomic DNA, especially repetitive sequences, can assume other types of structures such hairpins, Z-DNA, triplex DNA (HDNA) or tetrahelical DNA structures [1,2,3,4,5,6]. Impediments to normal DNA metabolic processes including transcription and replication imposed by such secondary DNA structures explain the correlation between repetitive sequence elements and elevated genome instability. Genomic instability at purine-rich GAANTTC repeats and CAGNCTG repeats, which can fold into threestranded H-DNA [7] and a slipped hairpin structure [8], forms the molecular basis of multiple neurodegenerative diseases, such as Freidreich’s Ataxia and Huntington’s disease, respectively. G4 DNA can be readily formed in solution by oligonucleotides containing multiple runs of guanines and by actively transcribed plasmid DNA [12,13]. The distribution of G4 motifs is highly concentrated at telomeres, rDNA loci, immunoglobulin heavy-chain switch regions, and G-rich minisatellites and significantly correlates with nucleosome-free regions and transcription start sites (TSSs) [10,11,18]. G4 motifs are mostly enriched in the regions flanking TSSs, which suggests that G4 DNA may be involved in transcriptional regulation [15,17]

Methods

Results

Discussion

Conclusion