Allison, R. M. | bioRxiv Science

bioRxiv2019

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

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 enables us to reveal the influence primary DNA sequence has upon Top2 cleavage--distinguishing canonical DNA double-strand breaks (DSBs) from a major population of DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

molecular biology↗

bioRxiv2019

Removal of Spo11 from meiotic DNA breaks in vitro but not in vivo by Tyrosyl DNA Phosphodiesterase 2

Meiotic recombination events are initiated by DNA double-strand breaks (DSBs) created by the topoisomerase-like protein, Spo11. Similar to type-II topoisomerases, Spo11 becomes covalently linked to the 5' ends generated on each side of the DSB. Whilst Spo11-oligos--the product of nucleolytic removal by Mre11--have been detected in a number of biological systems, the lifetime of the covalent Spo11-DSB precursor has not been systematically determined and may be subject to alternative processing reactions. Here we explore the activity of human Tyrosyl DNA Phosphodiesterase, TDP2, on Spo11-DSBs isolated from S. cerevisiae cells. We demonstrate that TDP2 can remove Spo11 from natural ssDNA-oligos, and dsDNA ends even when in the presence of excess competitor genomic DNA. Interestingly, TDP2-processed Spo11-DSBs are refractory to resection by Exo1, suggesting that ssDNA generated by Mre11 may be essential in vivo to facilitate resection-dependent HR at Spo11-DSBs even if TDP2 were active. Moreover, although TDP2 can remove Spo11 peptides in vitro, TDP2 was unable to remove Spo11 in vivo--unlike during the repair of topoisomerase-induced DNA lesions. These results suggest that Spo11-DNA, but not topoisomerase-DNA cleavage complexes, are inaccessible to the TDP2 enzyme, perhaps due to occlusion by higher order protein complexes resident at sites of meiotic recombination.

molecular biology↗