A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

William H Gittens,Holly Thomas,Tim J Cooper,Dominic J Johnson,Matthew J Neale,Rachal M Allison

doi:10.1038/s41467-019-12802-5

Abstract

DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

Highlights

DNA topoisomerases are required to resolve DNA topological stress
Ethanol-fixed meiotic S. cerevisiae cells were lysed in the absence of proteolysis using strong protein-denaturing detergent at 65 ̊C and extracted with a phenol/chloroform mixture to remove noncovalently bound protein, generating an aqueous phase enriched in total genomic DNA and putative Spo[11] covalent complex (CC)
Such purified genomic DNA was digested with PstI restriction enzyme, and passed through glass-fibre spin columns in the presence of a high salt buffer that promotes protein binding[22,23], but suppresses the binding of DNA not covalently linked to protein

Summary

Introduction

DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. The specific mechanisms by which this is accomplished vary considerably across the family, key aspects are shared: including single or double-strand DNA cleavage to form a transient covalent complex (CC), which allows alteration of the topology of the nucleic acid substrate prior to religation[2] These processes are essential but carry with them a significant risk to genome stability because the CC may be stabilised as a permanent proteinlinked DNA break by several physiological factors; such as the proximity of other DNA lesions, the collision of transcription and replication complexes, denaturation of the topoisomerase, or by the binding of small molecules that inhibit religation[1,3]. TOP2β apparently plays an important role in promoting transcriptional programmes associated with neuronal development[13,14], a function that cannot be supported by TOP2α

Methods

Results

Conclusion