Abstract
Topoisomerase 1 (Top1) removes transcription-associated helical stress to suppress G4-formation and its induced recombination at genomic loci containing guanine-run containing sequences. Interestingly, Top1 binds tightly to G4 structures, and its inhibition or depletion can cause elevated instability at these genomic 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 expression of Top1 cleavage-defective mutants but not a DNA-binding-defective mutant lead to significantly elevated instability at a G4-forming genomic locus. Elevated recombination rates were partly suppressed by their proteolytic removal 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 increase 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.
Highlights
Repetitive DNA sequences are hotspots of genomic instability [1]
Topoisomerase 1 (Top1) mutants used in the study Previously, we reported that the expression of catalytically dead Top1Y727F mutation results in a significant elevation of guanine quadruplex (G4)-associated genome instability over Top1-absence, indicating a gain of function by this catalytic mutant protein [28]
In order to determine the mechanism underlying such elevated G4-specific genome instability mediated by the Top1 mutant, we constructed two other yeast strains with TOP1 mutant alleles (Table 1). yTop1Y740Stop is analogous to the human Top1W763Stop mutant identified in a lung cancer patient treated with the CPT-derivative
Summary
Repetitive DNA sequences are hotspots of genomic instability [1]. The potential of these repetitive sequences to form non-B DNA structures that perturb replication and transcription exacerbates instability associated with them. For sequences containing guanine runs, non-B secondary DNA structures called G-quadruplexes (G4s) can arise when planar guanine tetrads made up of four guanine bases stabilized through Hoogsteen bonding stack upon one another [2, 3]. G4-formation usually requires the minimal sequence of GGGN1-7GGGN1-7GGGN1-7GGG, where N can be any of the DNA bases and comprises loops that extrude outward from the stacked guanine tetrads. The non-random genomic locations of G4-motifs indicate that these structures have roles in the regulation of important cellular processes like transcription and programmed recombination
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