scholarly journals The Role of Topoisomerase II in DNA Repair and Recombination in Arabidopsis thaliana

2021 ◽  
Vol 22 (23) ◽  
pp. 13115
Author(s):  
Marina Martinez-Garcia ◽  
Charles I. White ◽  
F. Chris. H. Franklin ◽  
Eugenio Sanchez-Moran
Keyword(s):  
Arabidopsis Thaliana ◽  
Dna Repair ◽  
Topoisomerase Ii ◽  
Model Organisms ◽  
Loop Formation ◽  
Dna Strands ◽  
D Loop ◽  
Endogenous Role ◽  
Strand Invasion

DNA entanglements and supercoiling arise frequently during normal DNA metabolism. DNA topoisomerases are highly conserved enzymes that resolve the topological problems that these structures create. Topoisomerase II (TOPII) releases topological stress in DNA by removing DNA supercoils through breaking the two DNA strands, passing a DNA duplex through the break and religating the broken strands. TOPII performs key DNA metabolic roles essential for DNA replication, chromosome condensation, heterochromatin metabolism, telomere disentanglement, centromere decatenation, transmission of crossover (CO) interference, interlock resolution and chromosome segregation in several model organisms. In this study, we reveal the endogenous role of Arabidopsis thaliana TOPII in normal root growth and cell cycle, and mitotic DNA repair via homologous recombination. Additionally, we show that the protein is required for meiotic DSB repair progression, but not for CO formation. We propose that TOPII might promote mitotic HR DNA repair by relieving stress needed for HR strand invasion and D-loop formation.

Role of topoisomerase inhibition and DNA repair mechanisms in the genotoxicity of alternariol and altertoxin-II

2013 ◽  
Vol 6 (3) ◽  
pp. 233-244 ◽  
Author(s):  
C. Tiessen ◽  
H. Gehrke ◽  
C. Kropat ◽  
C. Schwarz ◽  
S. Bächler ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Topoisomerase I ◽  
Topoisomerase Ii ◽  
Dna Lesions ◽  
Nuclear Antigen ◽  
Substantial Impact ◽  
Ht29 Cells ◽  
Repair Pathways

Alternariol (AOH) and altertoxin-II (ALTX-II) have been demonstrated to possess genotoxic properties. However, the underlying mechanisms of action have not been fully elucidated yet. AOH has recently been shown to act as a topoisomerase I and II poison, contributing to its genotoxic properties. The topoisomerase-specific repair factor tyrosyl-DNA-phosphodiesterase-1 (TDP1) is involved in the respective repair processes of damaged DNA induced by topoisomerase II poison. In the present study, we investigated the role of DNA repair pathways for the extent of DNA damage by AOH and addressed the question whether interference with topoisomerase II might play a role in the genotoxicity of ALTX-II. Under cell-free conditions, AOH and ALTX-II suppressed the activity of topoisomerase II at a comparable concentration range. In HT29 cells, AOH enhanced the level of covalent DNA-topoisomerase II complexes, thus acting as a topoisomerase poison in DNA damaging concentrations. In contrast, ALTX-II in genotoxic concentrations did not show any effect on the stability of these complexes, indicating that interference with topoisomerases does not play a relevant role in genotoxicity. The differences in genotoxic mechanisms seem to be reflected in the activation of p53. AOH was found to increase p53 phosphorylation in HT29 cells in DNA damaging concentrations. In contrast, incubation with ALTX-II did not affect p53 phosphorylation despite substantial increase in tail intensity in the comet assay, suggesting that the DNA lesions formed by ALTX-II are not detected by the DNA-repair machinery of HT29 cells. These results are supported by differences in persistence of DNA damage, still maintained after 24 h for ALTX-II but nearly vanished already after 3 h for AOH. Furthermore, microarray and qPCR analysis did not indicate any substantial impact of AOH on the transcription of key elements of DNA repair pathways. However, siRNA-approaches indicate that, in addition to TDP1, the expression of other elements of the DNA repair machinery exemplified by the 70 kDa Ku autoantigen and the proliferating cell nuclear antigen are relevant for AOH-mediated DNA damage.

Targeting DNA Repair Systems in Antitubercular Drug Development

2019 ◽  
Vol 26 (8) ◽  
pp. 1494-1505 ◽  
Author(s):  
Alina Minias ◽  
Anna Brzostek ◽  
Jarosław Dziadek
Keyword(s):  
Dna Repair ◽  
Protein Crystal ◽  
Model Organisms ◽  
Dna Repair Genes ◽  
Associated Proteins ◽  
Related Proteins ◽  
Repair Genes

Infections with Mycobacterium tuberculosis, the causative agent of tuberculosis, are difficult to treat using currently available chemotherapeutics. Clinicians agree on the urgent need for novel drugs to treat tuberculosis. In this mini review, we summarize data that prompts the consideration of DNA repair-associated proteins as targets for the development of new antitubercular compounds. We discuss data, including gene expression data, that highlight the importance of DNA repair genes during the pathogenic cycle as well as after exposure to antimicrobials currently in use. Specifically, we report experiments on determining the essentiality of DNA repair-related genes. We report the availability of protein crystal structures and summarize discovered protein inhibitors. Further, we describe phenotypes of available gene mutants of M. tuberculosis and model organisms Mycobacterium bovis and Mycobacterium smegmatis. We summarize experiments regarding the role of DNA repair-related proteins in pathogenesis and virulence performed both in vitro and in vivo during the infection of macrophages and animals. We detail the role of DNA repair genes in acquiring mutations, which influence the rate of drug resistance acquisition.

scholarly journals UAF1 DNA Binding Activity Is Critical for RAD51-Mediated Homologous DNA Pairing

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2497-2497
Author(s):  
Fengshan Liang ◽  
Adam S Miller ◽  
Carolilne Tang ◽  
Patrick Sung ◽  
Gary M. Kupfer
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Binding ◽  
Complex Formation ◽  
Binding Activity ◽  
Loop Formation ◽  
Dna Binding Activity ◽  
D Loop ◽  
In Cells ◽  
Dna Pairing

Background: In the Fanconi anemia (FA) DNA repair pathway, DNA damage induces the mono-ubiquitination of the FANCI-FANCD2 (ID2) heterodimer by the FA core complex through its inherent E3 ligase activity. The timely deubiquitination of ID2 by USP1-UAF1 deubiquitinase complex is also critically important for the FA DNA repair. UAF1 has a DNA binding activity, which is required for FANCD2 deubiquitination. UAF1 also enhances RAD51-mediated homologous DNA pairing in a manner that is dependent on complex formation with RAD51AP1. UAF1 deficient cells are impaired for DNA repair by homologous recombination (HR).The biochemical and cellular functions of UAF1 DNA binding activity in HR remain elusive. Methods:UAF1 wild type and DNA binding mutant proteins were purified and used to define its biochemical properties in HR. In vitroD-loop formation and synaptic complex assembly assay were performed to discover the DNA binding of UAF1 in RAD51 recombinase enhancement. U2OS-DR-GFP cell lines with impaired UAF1 or RAD51AP1DNA binding were generated to examine HR efficiency and DNA damage resistance. Results:UAF1 preferentially binds an HR-intermediate-like DNA substrate (D-loop, Fig.1). The DNA binding deficient mutant of UAF1 is unable to stimulate RAD51AP1 promotion of RAD51-mediated D-loop (Fig. 2) and the ability to recruit homologous DNA to form the presynaptic complex formation in HR (Fig. 3). In cells, the UAF1 DNA-binding mutant is compromised for the ability to repair DNA damage and to implement HR (Fig. 4). Such activity correlates with the ability to confer resistance to DNA cross linking agents such as mitomycin C (Fig. 4). The DNA binding of UAF1 and RAD51AP1 have a coordinated role in HR-directed DNA damage repair (Fig. 5). Conclusions: UAF1 DNA binding activity is indispensable for its function in enhancing RAD51-mediated homologous DNA pairing within the context of the UAF1-RAD51AP1 complex. UAF1 DNA binding deficiency causes DNA damage sensitivity and impairs HR efficiency in cells. Translational Applicability:Our findings reveal a critical role of UAF1 DNA binding in DNA repair and genome maintenance. The identification of UAF1's role in repair will enable targeted efforts to improve molecular approaches for FA therapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rdh54/Tid1 Inhibits Rad51-Rad54-Mediated D-loop Formation and Limits D-loop Length

2020 ◽  
Author(s):  
Shanaya Shital Shah ◽  
Stella Hartono ◽  
Aurèle Piazza ◽  
Vanessa Som ◽  
William Wright ◽  
...  
Keyword(s):  
Binding Sites ◽  
Loop Length ◽  
Loop Formation ◽  
Independent Manner ◽  
Potential Binding ◽  
D Loop ◽  
Strand Invasion ◽  
Type Switch ◽  
Dna Strand

ABSTRACTDisplacement loops (D-loops) are intermediates formed during homologous recombination that play a pivotal role in the fidelity of repair. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54 is also present in somatic cells where its function is less understood. While Rdh54 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54-mediated invasions. Here, we show that Rdh54 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54 uniquely restricts the lengths of Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast, whereas Rdh54 expression is suppressed in diploids. We propose that Rdh54 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54 acts as a physical roadblock to Rad54’s translocation activity, limiting D-loop formation and D-loop length.

scholarly journals Aberrant repair initiated by the adenine-DNA glycosylase does not play a role in UV-induced mutagenesis in Escherichia coli

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e6029 ◽  
Author(s):  
Caroline Zutterling ◽  
Aibek Mursalimov ◽  
Ibtissam Talhaoui ◽  
Zhanat Koshenov ◽  
Zhiger Akishev ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Dna Repair ◽  
Uv Irradiation ◽  
Genome Stability ◽  
Dna Duplexes ◽  
E Coli ◽  
Dna Strands ◽  
Induced Mutagenesis

Background DNA repair is essential to counteract damage to DNA induced by endo- and exogenous factors, to maintain genome stability. However, challenges to the faithful discrimination between damaged and non-damaged DNA strands do exist, such as mismatched pairs between two regular bases resulting from spontaneous deamination of 5-methylcytosine or DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved the mismatch-specific DNA glycosylases that can recognize and remove regular DNA bases in the mismatched DNA duplexes. The Escherichia coli adenine-DNA glycosylase (MutY/MicA) protects cells against oxidative stress-induced mutagenesis by removing adenine which is mispaired with 7,8-dihydro-8-oxoguanine (8oxoG) in the base excision repair pathway. However, MutY does not discriminate between template and newly synthesized DNA strands. Therefore the ability to remove A from 8oxoG•A mispair, which is generated via misincorporation of an 8-oxo-2′-deoxyguanosine-5′-triphosphate precursor during DNA replication and in which A is the template base, can induce A•T→C•G transversions. Furthermore, it has been demonstrated that human MUTYH, homologous to the bacterial MutY, might be involved in the aberrant processing of ultraviolet (UV) induced DNA damage. Methods Here, we investigated the role of MutY in UV-induced mutagenesis in E. coli. MutY was probed on DNA duplexes containing cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproduct (6–4PP). UV irradiation of E. coli induces Save Our Souls (SOS) response characterized by increased production of DNA repair enzymes and mutagenesis. To study the role of MutY in vivo, the mutation frequencies to rifampicin-resistant (RifR) after UV irradiation of wild type and mutant E. coli strains were measured. Results We demonstrated that MutY does not excise Adenine when it is paired with CPD and 6–4PP adducts in duplex DNA. At the same time, MutY excises Adenine in A•G and A•8oxoG mispairs. Interestingly, E. coli mutY strains, which have elevated spontaneous mutation rate, exhibited low mutational induction after UV exposure as compared to MutY-proficient strains. However, sequence analysis of RifR mutants revealed that the frequencies of C→T transitions dramatically increased after UV irradiation in both MutY-proficient and -deficient E. coli strains. Discussion These findings indicate that the bacterial MutY is not involved in the aberrant DNA repair of UV-induced DNA damage.

scholarly journals RAD52: Paradigm of Synthetic Lethality and New Developments

2021 ◽  
Vol 12 ◽  
Author(s):  
Matthew J. Rossi ◽  
Sarah F. DiDomenico ◽  
Mikir Patel ◽  
Alexander V. Mazin
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Cancer Cells ◽  
Synthetic Lethality ◽  
Single Strand ◽  
Cancer Therapies ◽  
Loop Formation ◽  
Dna Double Strand Breaks ◽  
New Findings ◽  
D Loop

DNA double-strand breaks and inter-strand cross-links are the most harmful types of DNA damage that cause genomic instability that lead to cancer development. The highest fidelity pathway for repairing damaged double-stranded DNA is termed Homologous recombination (HR). Rad52 is one of the key HR proteins in eukaryotes. Although it is critical for most DNA repair and recombination events in yeast, knockouts of mammalian RAD52 lack any discernable phenotypes. As a consequence, mammalian RAD52 has been long overlooked. That is changing now, as recent work has shown RAD52 to be critical for backup DNA repair pathways in HR-deficient cancer cells. Novel findings have shed light on RAD52’s biochemical activities. RAD52 promotes DNA pairing (D-loop formation), single-strand DNA and DNA:RNA annealing, and inverse strand exchange. These activities contribute to its multiple roles in DNA damage repair including HR, single-strand annealing, break-induced replication, and RNA-mediated repair of DNA. The contributions of RAD52 that are essential to the viability of HR-deficient cancer cells are currently under investigation. These new findings make RAD52 an attractive target for the development of anti-cancer therapies against BRCA-deficient cancers.

scholarly journals Rdh54/Tid1 inhibits Rad51-Rad54-mediated D-loop formation and limits D-loop length

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Shanaya Shital Shah ◽  
Stella Hartono ◽  
Aurèle Piazza ◽  
Vanessa Som ◽  
William Wright ◽  
...  
Keyword(s):  
Binding Sites ◽  
Loop Length ◽  
Loop Formation ◽  
Independent Manner ◽  
Potential Binding ◽  
D Loop ◽  
Strand Invasion ◽  
Type Switch ◽  
Dna Strand

Displacement loops (D-loops) are critical intermediates formed during homologous recombination. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54/Tid1 is also present in somatic cells where its function is less understood. While Rdh54/Tid1 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54. Here, we show that Rdh54/Tid1 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54/Tid1 uniquely restricts the length of Rad51-Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast. We propose that Rdh54/Tid1 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54/Tid1 acts as a physical roadblock to Rad54 translocation, limiting D-loop formation and D-loop length.

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