Topoisomerase II contributes to DNA secondary structure-mediated double-stranded breaks

Abstract

<jats:title>Abstract</jats:title> <jats:p>DNA double-stranded breaks (DSBs) trigger human genome instability, therefore identifying what factors contribute to DSB induction is critical for our understanding of human disease etiology. Using an unbiased, genome-wide approach, we found that genomic regions with the ability to form highly stable DNA secondary structures are enriched for endogenous DSBs in human cells. Human genomic regions predicted to form non-B-form DNA induced gross chromosomal rearrangements in yeast and displayed high indel frequency in human genomes. The extent of instability in both analyses is in concordance with the structure forming ability of these regions. We also observed an enrichment of DNA secondary structure-prone sites overlapping transcription start sites (TSSs) and CCCTC-binding factor (CTCF) binding sites, and uncovered an increase in DSBs at highly stable DNA secondary structure regions, in response to etoposide, an inhibitor of topoisomerase II (TOP2) re-ligation activity. Importantly, we found that TOP2 deficiency in both yeast and human leads to a significant reduction in DSBs at structure-prone loci, and that sites of TOP2 cleavage have a greater ability to form highly stable DNA secondary structures. This study reveals a direct role for TOP2 in generating secondary structure-mediated DNA fragility, advancing our understanding of mechanisms underlying human genome instability.</jats:p>

Short-range template switching in great ape genomes explored using pair hidden Markov models

“…In the ancestral sequences, the smaller decrease in observed free energy around ① is reflective of pre-event potential for structural formation in a subset of events, suggesting that some events may involve hairpin-mediated quasipalindrome-to-palindrome conversion as in the original mechanism proposed for bacteria [ 22 ]. Regardless of ancestral stability, the spontaneous creation of sequence regions capable of forming stable secondary structures is of note, as small regions of stable structure play a role in several biological processes [ 42 , 43 ], and regions of similarly stable structure can cause fork collapse, DSB formation and trigger genome instability [ 44 , 45 ].…”

“…We calculated minor groove width, helical twist and propeller twist in these regions, as well as for 100,000 uniform random sampled 1001nt sequences from across all GRCh38 chromosomes, using the DNAShapeR package [ 45 ], which is based on the method of [ 69 ] for predicting DNA structural information. This approach utilises a pentamer sliding window to calculate each feature as determined through Monte Carlo simulations, accounting for sequence context of the focal nucleotide within the window.…”

Transcription-associated topoisomerase 2α (TOP2A) activity is a major effector of cytotoxicity induced by G-quadruplex ligands

“…Genome-wide analyses of G4 motifs in human cells indicate that these structures are highly correlated with RNA-Pol II pausing sites and R-loop-forming regions, two different factors promoting RNA-Pol II arrests and transcription-dependent DNA breaks ( Puget et al, 2019 ; Chen et al, 2017 ; Eddy et al, 2011 ). Additionally, the formation of highly stable DNA secondary structures, such as G4, has been shown to promote the formation of DNA topoisomerase 2 (TOP2)-mediated DNA breaks ( Szlachta et al, 2020 ). Interestingly, recent studies demonstrate a major contribution of TOP2 activity in the generation of DSBs in highly transcribed genes ( Canela et al, 2019 ; Gittens et al, 2019 ; Gothe et al, 2019 ).…”

Short-range template switching in great ape genomes explored using a pair hidden Markov model

“…In the ancestral sequences, the smaller decrease in observed free energy around ① is reflective of pre-event potential for structural formation in a subset of events, suggesting that some events may involve hairpin-mediated quasipalindrome-to-palindrome conversion as in the original mechanism proposed for bacteria (22). Regardless of ancestral stability, the spontaneous creation of sequence regions capable of forming stable secondary structures is of note, as small regions of stable structure play a role in several biological processes (39, 40), and regions of similarly stable structure can cause fork collapse, DSB formation and trigger genome instability (41, 42).…”