An oxidized abasic lesion inhibits base excision repair leading to DNA strand breaks in a trinucleotide repeat tract

Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl2) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms.

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Trinucleotide repeat instability via DNA base excision repair

DNA Repair ◽

10.1016/j.dnarep.2020.102912 ◽

2020 ◽

Vol 93 ◽

pp. 102912

Author(s):

Yanhao Lai ◽

Jill M. Beaver ◽

Eduardo Laverde ◽

Yuan Liu

Keyword(s):

Base Excision Repair ◽

Trinucleotide Repeat ◽

Excision Repair ◽

Repeat Instability ◽

Dna Base Excision Repair ◽

Dna Base ◽

Base Excision

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R-loops promote trinucleotide repeat deletion through DNA base excision repair enzymatic activities

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014161 ◽

2020 ◽

Vol 295 (40) ◽

pp. 13902-13913

Author(s):

Eduardo E. Laverde ◽

Yanhao Lai ◽

Fenfei Leng ◽

Lata Balakrishnan ◽

Catherine H. Freudenreich ◽

...

Keyword(s):

Base Excision Repair ◽

Trinucleotide Repeat ◽

Excision Repair ◽

Enzymatic Activities ◽

Cag Repeat ◽

Abasic Site ◽

Dna Damage And Repair ◽

Dna Base Excision Repair ◽

Treatment And Prevention ◽

Base Excision

Trinucleotide repeat (TNR) expansion and deletion are responsible for over 40 neurodegenerative diseases and associated with cancer. TNRs can undergo somatic instability that is mediated by DNA damage and repair and gene transcription. Recent studies have pointed toward a role for R-loops in causing TNR expansion and deletion, and it has been shown that base excision repair (BER) can result in CAG repeat deletion from R-loops in yeast. However, it remains unknown how BER in R-loops can mediate TNR instability. In this study, using biochemical approaches, we examined BER enzymatic activities and their influence on TNR R-loops. We found that AP endonuclease 1 incised an abasic site on the nontemplate strand of a TNR R-loop, creating a double-flap intermediate containing an RNA:DNA hybrid that subsequently inhibited polymerase β (pol β) synthesis of TNRs. This stimulated flap endonuclease 1 (FEN1) cleavage of TNRs engaged in an R-loop. Moreover, we showed that FEN1 also efficiently cleaved the RNA strand, facilitating pol β loop/hairpin bypass synthesis and the resolution of TNR R-loops through BER. Consequently, this resulted in fewer TNRs synthesized by pol β than those removed by FEN1, thereby leading to repeat deletion. Our results indicate that TNR R-loops preferentially lead to repeat deletion during BER by disrupting the balance between the addition and removal of TNRs. Our discoveries open a new avenue for the treatment and prevention of repeat expansion diseases and cancer.

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DNA Repair Functions That Control Sensitivity to Topoisomerase-Targeting Drugs

Eukaryotic Cell ◽

10.1128/ec.3.1.82-90.2004 ◽

2004 ◽

Vol 3 (1) ◽

pp. 82-90 ◽

Cited By ~ 39

Author(s):

Mobeen Malik ◽

John L. Nitiss

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Topoisomerase I ◽

Topoisomerase Ii ◽

Excision Repair ◽

Dna Strand Breaks ◽

Dna Topology ◽

Strand Breaks ◽

Wide Range ◽

Dna Strand

ABSTRACT DNA topoisomerases play critical roles in a wide range of cellular processes by altering DNA topology to facilitate replication, transcription, and chromosome segregation. Topoisomerases alter DNA topology by introducing transient DNA strand breaks that involve a covalent protein DNA intermediate. Many agents have been found to prevent the religation of DNA strand breaks induced by the enzymes, thereby converting the enzymes into DNA-damaging agents. Repair of the DNA damage induced by topoisomerases is significant in understanding drug resistance arising following treatment with topoisomerase-targeting drugs. We have used the fission yeast Schizosaccharomyces pombe to identify DNA repair pathways that are important for cell survival following drug treatment. S. pombe strains carrying mutations in genes required for homologous recombination such as rad22A or rad32 (homologues of RAD52 and MRE11) are hypersensitive to drugs targeting either topoisomerase I or topoisomerase II. In contrast to results observed with Saccharomyces cerevisiae, S. pombe strains defective in nucleotide excision repair are also hypersensitive to topoisomerase-targeting agents. The loss of DNA replication or DNA damage checkpoints also sensitizes cells to both topoisomerase I and topoisomerase II inhibitors. Finally, repair genes (such as the S. pombe rad8+ gene) with no obvious homologs in other systems also play important roles in causing sensitivity to topoisomerase drugs. Since the pattern of sensitivity is distinct from that seen with other systems (such as the S. cerevisiae system), our results highlight the usefulness of S. pombe in understanding how cells deal with the unique DNA damage induced by topoisomerases.

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Prevention of base excision repair by TRC102 (methoxyamine) potentiates the anti-tumor activity of pemetrexed in vitro and in vivo

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.13005 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 13005-13005 ◽

Cited By ~ 2

Author(s):

L. Liu ◽

A. Bulgar ◽

J. Donze ◽

B. J. Adams ◽

C. P. Theuer ◽

...

Keyword(s):

Excision Repair ◽

Alkylating Agents ◽

Phase 1 ◽

Dna Strand Breaks ◽

Strand Breaks ◽

Ap Sites ◽

Dna Strand ◽

Anti Tumor Activity

13005 Background: TRC102 (methoxyamine) reverses resistance to alkylating agents by inhibiting base excision repair (BER; a mechanism of DNA repair), thereby increasing DNA strand breaks and potentiating the anti-tumor activity of alkylating agents without additional toxicity, Based on these data, TRC102 is currently being studied in combination with temozolomide in a phase 1 trial. We hypothesized that inhibition of BER by TRC102 would also increase DNA strand breaks and improve the anti-tumor activity of anti-metabolite chemotherapeutics, including pemetrexed, because these agents also produce AP sites that are recognized and repaired by BER. Methods: Pemetrexed- induced AP sites and BER inhibition was quantified using an apurinic/apyrimidinic (AP) site assay in vitro. Single and double DNA strand breaks were quantified by the Comet assay in vitro and anti-tumor activity was assessed in an in vivo xenograft study of subcutaneously implanted H460 human lung cancer cells. Results: Pemetrexed induced and TRC102 reduced the number of available AP sites in pemetrexed- treated H460 cells (by 60–80%), indicating successful inhibition of BER. TRC102 treatment increased DNA strand breaks in pemetrexed-treated H460 cells (2 fold increase versus treatment with pemetrexed alone). Premetrexed treatment alone and in combination with TRC 102 delayed tumor growth in vivo (tumor growth delay of 4.7 days in the 150 mg/m2 pemetrexed alone group, 5.7 days in the 150 mg/m2 pemetrexed + 2 mg/m2 TRC102 group and 6.9 days in the 150 mg/m2 pemetrexed + 4 mg/m2 TRC102 group); in vivo systemic toxicity was not increased. TRC102 alone had no effect in vitro or in vivo. Conclusions: TRC102 effectively inhibits BER in lung cancer cells treated with pemetrexed. Inhibition of DNA repair by TRC102 results in an increase in DNA strand breaks and improved anti-tumor activity versus treatment with pemetrexed alone. Given its preclinical efficacy and safety profile, study of TRC102 combined with pemetrexed in a phase 1 trial is warranted. No significant financial relationships to disclose.

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Trinucleotide Repeat Instability is Modulated by DNA Base Lesions and DNA Base Excision Repair

10.25148/etd.fidc001178 ◽

2016 ◽

Author(s):

Jill M Beaver

Keyword(s):

Base Excision Repair ◽

Trinucleotide Repeat ◽

Excision Repair ◽

Repeat Instability ◽

Dna Base Excision Repair ◽

Dna Base ◽

Base Excision

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PARP1 and PARP2 stabilise replication forks at base excision repair intermediates through Fbh1-dependent Rad51 regulation

10.1101/243071 ◽

2018 ◽

Author(s):

George E. Ronson ◽

Ann Liza Piberger ◽

Martin R. Higgs ◽

Anna L. Olsen ◽

Grant S. Stewart ◽

...

Keyword(s):

Base Excision Repair ◽

Damage Tolerance ◽

Excision Repair ◽

Replication Forks ◽

Strand Breaks ◽

Single Strand Breaks ◽

Dna Base ◽

Repair Mechanisms ◽

Base Excision

AbstractPARP1 regulates the repair of DNA single strand breaks (SSBs) generated directly, or during base excision repair (BER). However, the role of PARP2 in these and other repair mechanisms is unknown. Here, we report a requirement for PARP2 in stabilising replication forks that encounter BER intermediates through Fbh1-dependent regulation of Rad51. Whilst PARP2 is dispensable for tolerance of cells to SSBs or homologous recombination dysfunction, it is redundant with PARP1 in BER. Therefore, combined disruption of PARP1 and PARP2 leads to defective BER, resulting in elevated levels of replication associated DNA damage due to an inability to stabilise Rad51 at damaged replication forks and prevent uncontrolled DNA resection. Together, our results demonstrate how PARP1 and PARP2 regulate two independent, but intrinsically linked aspects of DNA base damage tolerance by promoting BER directly, and through stabilising replication forks that encounter BER intermediates.

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Induction of CAF-1 Expression in Response to DNA Strand Breaks in Quiescent Human Cells

Molecular and Cellular Biology ◽

10.1128/mcb.26.5.1839-1849.2006 ◽

2006 ◽

Vol 26 (5) ◽

pp. 1839-1849 ◽

Cited By ~ 30

Author(s):

Arman Nabatiyan ◽

Dávid Szüts ◽

Torsten Krude

Keyword(s):

Excision Repair ◽

Dna Strand Breaks ◽

Significant Loss ◽

Human Cells ◽

Dna Breaks ◽

Strand Breaks ◽

Quiescent Cells ◽

Dna Strand

ABSTRACT Genome stability in eukaryotic cells is maintained through efficient DNA damage repair pathways, which have to access and utilize chromatin as their natural template. Here we investigate the role of chromatin assembly factor 1 (CAF-1) and its interacting protein, PCNA, in the response of quiescent human cells to DNA double-strand breaks (DSBs). The expression of CAF-1 and PCNA is dramatically induced in quiescent cells upon the generation of DSBs by the radiomimetic drug bleocin (a bleomycin compound) or by ionizing radiation. This induction depends on DNA-PK. CAF-1 and PCNA are recruited to damaged chromatin undergoing DNA repair of single- and double-strand DNA breaks by the base excision repair and nonhomologous end-joining pathways, respectively, in the absence of extensive DNA synthesis. CAF-1 prepared from repair-proficient quiescent cells after induction by bleocin mediates nucleosome assembly in vitro. Depletion of CAF-1 by RNA interference in bleocin-treated quiescent cells in vivo results in a significant loss of cell viability and an accumulation of DSBs. These results support a novel and essential role for CAF-1 in the response of quiescent human cells to DSBs, possibly by reassembling chromatin following repair of DNA strand breaks.

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