Cleavage-defective Topoisomerase I mutants sharply increase G-quadruplex-associated genomic instability

Abstract

Topoisomerase 1 (Top1) removes transcription-associated helical stress to sup-press G4-formation and its induced recombination at genomic loci containing guanine-run containing sequences. Interestingly, Top1 binds tightly to G4 struc-tures, and its inhibition or depletion can cause elevated instability at these ge-nomic loci. Top1 is targeted by the widely used anti-cancer chemotherapeutic camptothecin (CPT) and its derivatives, which stabilize Top1 covalently attached on a DNA nick and prevent the re-ligation step. Here we investigated how CPT-resistance conferring Top1 mutants, which emerge in cancer patients and cells treated with CPT, affect G4-induced genomic instability in S. cerevisiae. We found that Top1 mutants form stable complexes with G4 DNA and that expres-sion of Top1 cleavage-defective mutants but not a DNA-binding-defective mu-tant lead to significantly elevated instability at a G4-forming genomic locus. Elevated recombination rates were partly suppressed by their proteolytic re-moval by SPRTN homolog Wss1 SUMO-dependent metalloprotease in vivo. Furthermore, interaction between G4-DNA binding protein Nsr1, a homolog to clinically-relevant human nucleolin, and Top1 mutants lead to a synergistic in-crease in G4-associated recombination. These results in the yeast system are strengthened by our cancer genome data analyses showing that functionally detrimental mutations in Top1 correlate with an enrichment of mutations at G4 motifs. Our collective experimental and computational findings point to cooperative binding of Top1 cleavage-defective mutants and Nsr1 as promoting DNA replication blockage and exacerbating genomic instability at G4-motifs, thus complicating patient treatment.

Keywords

• g-quadruplex
• topoisomerase
• transcription
• recombination
• genome instability