Exploring the removal of Spo11 and topoisomerases from DNA breaks in S. cerevisiae by human Tyrosyl DNA Phosphodiesterase 2

• Published in: DNA repair Vol. 142; p. 103757
• Main Authors: Johnson, Dominic, Allison, Rachal M., Cannavo, Elda, Cejka, Petr, Harper, Jon A., Neale, Matthew J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2024
• Subjects: DNA Breaks, Double-Stranded; DNA Repair; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - metabolism; Double-strand break; Endodeoxyribonucleases - genetics; Endodeoxyribonucleases - metabolism; Exodeoxyribonucleases - genetics; Exodeoxyribonucleases - metabolism; Humans; Meiosis; Phosphoric Diester Hydrolases - metabolism; Recombination; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Spo11; TDP2; Topoisomerase; Topoisomerase; Recombination; Meiosis; Spo11; TDP2; Double-strand break
• ISSN: 1568-7864; 1568-7856; 1568-7856
• DOI: 10.1016/j.dnarep.2024.103757

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) created by Spo11, a type-II topoisomerase-like protein that becomes covalently linked to DSB ends. Whilst Spo11 oligos—the products of nucleolytic removal by Mre11—have been detected in several organisms, the lifetime of the covalent Spo11-DSB precursor has not been determined and may be subject to alternative processing. Here, we explore the activity of human Tyrosyl DNA Phosphodiesterase, TDP2—a protein known to repair DNA ends arising from abortive topoisomerase activity—on Spo11 DSBs isolated from S. cerevisiae cells. We demonstrate that TDP2 can remove Spo11 peptides from ssDNA oligos and dsDNA ends even in the presence of competitor genomic DNA. Interestingly, TDP2-processed DSB ends are refractory to resection by Exo1, suggesting that ssDNA generated by Mre11 may be essential in vivo to facilitate HR at Spo11 DSBs even if TDP2 were active. Moreover, although TDP2 can remove Spo11 peptides in vitro, TDP2 expression in meiotic cells was unable to remove Spo11 in vivo—contrasting its ability to aid 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 at sites of meiotic recombination. •Mammalian TDP2 removes Spo11 peptides from DNA ends in vitro.•TDP2-processed ends are refractory to resection by Exo1 in vitro.•Ectopic expression of TDP2 is unable to remove Spo11 from DSB ends in vivo.