Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Sep 16;26(18):9017.
doi: 10.3390/ijms26189017.

Mutations and Recombination at G4 DNA-Forming Sequences Exacerbated by CPT-Resistant Mutant Topoisomerase 1 Is Dependent on SUMOylation

Shivani Singh  1 , Xinji Zhu  2 , Nayun Kim  1   2
Affiliations

Mutations and Recombination at G4 DNA-Forming Sequences Exacerbated by CPT-Resistant Mutant Topoisomerase 1 Is Dependent on SUMOylation

Shivani Singh et al. Int J Mol Sci. .

Abstract

Topoisomerase 1 (Top1) removes transcription-related helical torsions and thus plays an important role in preventing genome instability instigated by the formation of non-canonical DNA secondary structures. The genetically tractable Saccharomyces cerevisiae model proved effective in defining the critical function of Top1 to prevent recombination and chromosomal rearrangement at G4-forming genomic loci and studying the human cancer-associated Top1 mutants through the expression of analogous yeast mutants. We previously showed that cleavage-defective Top1 mutants strongly elevate the rate of recombination at G4 DNA, which involves binding to G4 DNA and interaction with the protein nucleolin (Nsr1 in yeast). Here, we further explored the mechanism of genome instability induced by the yeast Top1Y740* mutant, analogous to the human Top1W765Stop mutant conferring resistance to CPT. We show that yTop1Y740* elevates duplications as well as recombination specifically at G4-forming sequences. Interestingly, SUMOylation of yTop1Y740*, which does not affect the G4 DNA-binding or Nsr1-interaction by this mutant, is necessary for such elevated G4-specific genome instability. Many tumors with mutations at the C-terminal residues of Top1, particularly W765, have significantly high G4-associated mutations, underscoring the importance of further investigation into how SUMOylation affects the function of these Top1 mutants at G4 DNA.

Keywords: G4 DNA; genome instability; topoisomerase 1.

PubMed Disclaimer

Conflict of interest statement

All authors declare that there are no conflicts of interest.

Figures

Figure 1
Figure 1
Rates of mutation and recombination in Top1Y740*-expressing cells. (A). The rates of deletion mutations at pTET-LYS2-AG4 reporter in rnh1∆ rnh201∆ backgrounds with the indicated Top1 alleles. (B). The rates of canavanine resistance mutations in the yeast strains with indicated Top1 alleles. (C). The rates of recombination at the pTET-LYS2-SmuR reporter with the C-run-containing strand in the non-transcribed strand orientation. (D). The rates of recombination at the pTET-LYS2-SmuF reporter with the G4-forming sequence in the non-transcribed strand orientation. (E). The rates of mutations at the GCA-repeat-containing reporter. (F). The rates of mutations at the pGAL-DIP2C reporter. (A–F): Error bars indicate 95% confidence intervals).
Figure 2
Figure 2
Mutation spectra at the pGAL-DIP2C reporter in top1∆ and Top1Y740*-expressing cells. The sequence of DIP2C inserted into the pGAL-DIP2C reporter is shown in bold with the guanine runs indicated in red. The spectrum of mutants sequenced are shown. The locations of duplicated and deleted sequences are marked by the arrows and insertion mutations are indicated with +. When the same mutant sequences are identified multiple times, they are indicated by the numbers in (). 47 (A) and 46 (B) mutants are sequenced from top1∆ and Top1Y740*-expressing strains, respectively.
Figure 3
Figure 3
The effect of eliminating SUMOylation of Top1Y740* on the rates of recombination at G4. (A). SUMO pull-down assay. At Top1 residues 65, 91, and 92, wildtype or mutated amino acid sequence are K or R, respectively. SUMO-modified, upshifted bands are indicated with a red asterisk. Cell extracts used in experiments are from strains in wss1∆ backgrounds and expressing Top1 tagged with 3XFLAG at C-term and Smt3 tagged with 7XHis at N-term. Pull-down of SUMO-modified proteins were carried out using Ni+ beads. Proteins detected by Western blotting with α-FLAG-HRP antibody. Top panel—pull-down samples (IP); bottom panel—inputs (IN). (B). Recombination rates at the G4-containing pTET-LYS2-SmuF reporter in cells expressing wildtype, Top1S733E, or Top1Y740*. Blue bars are rates from wildtype or mutant Top1 with K65, K91, and K92. Orange bars are rates from wildtype or mutant Top1 with K65R, K91R, and K92R mutations. Error bars indicate 95% confidence intervals. (C). SUMO pull-down assay. Pull-down experiments were carried out with wildtype or indicated Top1 mutant-expressing cells in either SIZ1 SIZ2 or siz1∆ siz2∆ backgrounds as indicated. Other details are same as (A). (D). Recombination rates at the G4-containing pTET-LYS2-SmuF reporter in cells expressing indicated Top1 alleles in either SIZ1 SIZ2 or siz1∆ siz2∆ backgrounds.
Figure 4
Figure 4
The effect of eliminating SUMOylation of Top1Y740* on G4 DNA binding. (A). The sequences and G4 Hunter scores for oligos used for the oligo pull-down assay. (B,C). Yeast whole-cell lysates from the indicated strains expressing WT or mutant Top1 proteins tagged with 3XFLAG were incubated with G4 or MUT biotinylated oligos and pulled down with streptavidin magnetic beads. Western blots of input and pull-down fractions were probed with an α-FLAG-HRP antibody (Sigma). Input and pull-down samples from wildtype Top1 in a SIZ1 SIZ2 background, wildtype Top1 in a siz1∆ siz2∆ background, and Top1K65R, K91R, K92R mutants in a SIZ1 SIZ2 background are shown in (B). Input and pull-down samples from Top1Y740* in a SIZ1 SIZ2 background, Top1Y740* in a siz1∆ siz2∆ background, and Top1Y740*, K65R, K91R, K92R mutants in a SIZ1 SIZ2 background are shown in (C).
Figure 5
Figure 5
The effect of eliminating SUMOylation of Top1Y740* on Nsr1 interaction. Co-immunoprecipitation (co-IP) experiments conducted with whole-cell extracts from vtc4∆ yeast strains expressing Top1-3XFLAG and Nsr1-6XHA. αFLAG antibody-coated agarose beads were used in pull-down. (A,B) are from one of three independent experiments carried out. The blots from the other two experiments are in Figure S1. Blots were probed with either αFLAG (A) or αHA (B) antibodies. (C). ImageJ v1.51 was used for the quantification of bands. Error bars represent standard deviations calculated from three independent experiments carried out. p-values were calculated using Student t-test. NS *—0.90, NS **—0.82, and NS ***—0.39.
See this image and copyright information in PMC

References

    1. Pommier Y., Leo E., Zhang H., Marchand C. DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem. Biol. 2010;17:421–433. doi: 10.1016/j.chembiol.2010.04.012. - DOI - PMC - PubMed
    1. Kim N., Jinks-Robertson S. Guanine repeat-containing sequences confer transcription-dependent instability in an orientation-specific manner in yeast. DNA Repair. 2011;10:953–960. doi: 10.1016/j.dnarep.2011.07.002. - DOI - PMC - PubMed
    1. Pavlova A.V., Kubareva E.A., Monakhova M.V., Zvereva M.I., Dolinnaya N.G. Impact of G-Quadruplexes on the Regulation of Genome Integrity, DNA Damage and Repair. Biomolecules. 2021;11:1284. doi: 10.3390/biom11091284. - DOI - PMC - PubMed
    1. Rider S.D., Jr., Gadgil R.Y., Hitch D.C., Damewood F.J.T., Zavada N., Shanahan M., Alhawach V., Shrestha R., Shin-Ya K., Leffak M. Stable G-quadruplex DNA structures promote replication-dependent genome instability. J. Biol. Chem. 2022;298:101947. doi: 10.1016/j.jbc.2022.101947. - DOI - PMC - PubMed
    1. Sato K., Knipscheer P. G-quadruplex resolution: From molecular mechanisms to physiological relevance. DNA Repair. 2023;130:103552. doi: 10.1016/j.dnarep.2023.103552. - DOI - PubMed

MeSH terms

Substances

LinkOut - more resources