A base-repair based electrochemiluminescent genotoxicity sensor that detects abasic sites in double-stranded DNA films

2020 ◽  
Vol 56 (83) ◽  
pp. 12558-12561
Author(s):  
Dong-Mei Wang ◽  
Jia Jia ◽  
Rong-Fu Huang ◽  
Xinfeng Zhang
Keyword(s):  
Abasic Sites ◽  
Double Stranded Dna ◽  
Ap Sites ◽  
Dna Films ◽  
Damaged Dna

An ECL-based genotoxicity sensor with the ability to identify the missed bases at AP sites in damaged DNA is developed.

Thermoplastic Nanofluidic Devices for Identifying Abasic Sites in Single DNA Molecules

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Steven A Soper ◽  
Swarnagowri Vaidyanathan ◽  
Franklin Uba ◽  
Bo Hu ◽  
David Kaufman ◽  
...  
Keyword(s):  
Dna Damage ◽  
Therapeutic Efficacy ◽  
Double Strand Breaks ◽  
Dna Molecules ◽  
Strand Breaks ◽  
Single Dna Molecules ◽  
Abasic Sites ◽  
Nanofluidic Devices ◽  
Ap Sites

DNA damage can take many forms such as double-strand breaks and/or the formation of abasic (apurinic/apyrimidinic; AP) sites. The presence of AP sites can be used to determine therapeutic efficacy...

scholarly journals Abasic Sites in the Transcribed Strand of Yeast DNA Are Removed by Transcription-Coupled Nucleotide Excision Repair

2010 ◽  
Vol 30 (13) ◽  
pp. 3206-3215 ◽  
Author(s):  
Nayun Kim ◽  
Sue Jinks-Robertson
Keyword(s):  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Genome Integrity ◽  
Genetic Studies ◽  
Abasic Sites ◽  
Dna And Rna ◽  
Nucleotide Excision ◽  
Ap Sites ◽  
Base Excision ◽  
Ap Site

ABSTRACT Abasic (AP) sites are potent blocks to DNA and RNA polymerases, and their repair is essential for maintaining genome integrity. Although AP sites are efficiently dealt with through the base excision repair (BER) pathway, genetic studies suggest that repair also can occur via nucleotide excision repair (NER). The involvement of NER in AP-site removal has been puzzling, however, as this pathway is thought to target only bulky lesions. Here, we examine the repair of AP sites generated when uracil is removed from a highly transcribed gene in yeast. Because uracil is incorporated instead of thymine under these conditions, the position of the resulting AP site is known. Results demonstrate that only AP sites on the transcribed strand are efficient substrates for NER, suggesting the recruitment of the NER machinery by an AP-blocked RNA polymerase. Such transcription-coupled NER of AP sites may explain previously suggested links between the BER pathway and transcription.

scholarly journals Molecular basis of abasic site sensing in single-stranded DNA by the SRAP domain of E. coli yedK

2019 ◽  
Vol 47 (19) ◽  
pp. 10388-10399 ◽  
Author(s):  
Na Wang ◽  
Hongyu Bao ◽  
Liu Chen ◽  
Yanhong Liu ◽  
Yue Li ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Substrate Binding ◽  
Specific Substrate ◽  
Abasic Site ◽  
E Coli ◽  
Abasic Sites ◽  
Ap Sites ◽  
Ap Site

Abstract HMCES and yedK were recently identified as sensors of abasic sites in ssDNA. In this study, we present multiple crystal structures captured in the apo-, nonspecific-substrate-binding, specific-substrate-binding, and product-binding states of yedK. In combination with biochemical data, we unveil the molecular basis of AP site sensing in ssDNA by yedK. Our results indicate that yedK has a strong preference for AP site-containing ssDNA over native ssDNA and that the conserved Glu105 residue is important for identifying AP sites in ssDNA. Moreover, our results reveal that a thiazolidine linkage is formed between yedK and AP sites in ssDNA, with the residues that stabilize the thiazolidine linkage important for the formation of DNA-protein crosslinks between yedK and the AP sites. We propose that our findings offer a unique platform to develop yedK and other SRAP domain-containing proteins as tools for detecting abasic sites in vitro and in vivo.

scholarly journals Response of Xenopus Cds1 in Cell-free Extracts to DNA Templates with Double-stranded Ends

2000 ◽  
Vol 11 (5) ◽  
pp. 1535-1546 ◽  
Author(s):  
Zijian Guo ◽  
William G. Dunphy
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Molecules ◽  
Dna Templates ◽  
Cell Cycle Delay ◽  
Checkpoint Kinases ◽  
Double Stranded Dna ◽  
Egg Extracts ◽  
Dna Ends ◽  
Damaged Dna

Although homologues of the yeast checkpoint kinases Cds1 and Chk1 have been identified in various systems, the respective roles of these kinases in the responses to damaged and/or unreplicated DNA in vertebrates have not been delineated precisely. Likewise, it is largely unknown how damaged DNA and unreplicated DNA trigger the pathways that contain these effector kinases. We report that XenopusCds1 (Xcds1) is phosphorylated and activated by the presence of some simple DNA molecules with double-stranded ends in cell-freeXenopus egg extracts. Xcds1 is not affected by aphidicolin, an agent that induces DNA replication blocks. In contrast,Xenopus Chk1 (Xchk1) responds to DNA replication blocks but not to the presence of double-stranded DNA ends. Immunodepletion of Xcds1 (and/or Xchk1) from egg extracts did not attenuate the cell cycle delay induced by double-stranded DNA ends. These results imply that the cell cycle delay triggered by double-stranded DNA ends either does not involve Xcds1 or uses a factor(s) that can act redundantly with Xcds1.

scholarly journals Role of Double-Stranded DNA Translocase Activity of Human HLTF in Replication of Damaged DNA

2009 ◽  
Vol 30 (3) ◽  
pp. 684-693 ◽  
Author(s):  
András Blastyák ◽  
Ildikó Hajdú ◽  
Ildikó Unk ◽  
Lajos Haracska
Keyword(s):  
Translesion Synthesis ◽  
Dna Lesions ◽  
Template Switching ◽  
Replication Forks ◽  
Double Stranded Dna ◽  
Fork Regression ◽  
Dna Translocase ◽  
Damaged Dna

ABSTRACT Unrepaired DNA lesions can block the progression of the replication fork, leading to genomic instability and cancer in higher-order eukaryotes. In Saccharomyces cerevisiae, replication through DNA lesions can be mediated by translesion synthesis DNA polymerases, leading to error-free or error-prone damage bypass, or by Rad5-mediated template switching to the sister chromatid that is inherently error free. While translesion synthesis pathways are highly conserved from yeast to humans, very little is known of a Rad5-like pathway in human cells. Here we show that a human homologue of Rad5, HLTF, can facilitate fork regression and has a role in replication of damaged DNA. We found that HLTF is able to reverse model replication forks, a process which depends on its double-stranded DNA translocase activity. Furthermore, from analysis of isolated dually labeled chromosomal fibers, we demonstrate that in vivo, HLTF promotes the restart of replication forks blocked at DNA lesions. These findings suggest that HLTF can promote error-free replication of damaged DNA and support a role for HLTF in preventing mutagenesis and carcinogenesis, providing thereby for its potential tumor suppressor role.

scholarly journals Evidence for the Involvement of Nucleotide Excision Repair in the Removal of Abasic Sites in Yeast

2000 ◽  
Vol 20 (10) ◽  
pp. 3522-3528 ◽  
Author(s):  
Carlos A. Torres-Ramos ◽  
Robert E. Johnson ◽  
Louise Prakash ◽  
Satya Prakash
Keyword(s):  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Repair Process ◽  
Dna Glycosylases ◽  
Abasic Sites ◽  
Skin Cancers ◽  
Nucleotide Excision ◽  
Progressive Neurodegeneration ◽  
Ap Sites ◽  
Base Excision

ABSTRACT In eukaryotes, DNA damage induced by ultraviolet light and other agents which distort the helix is removed by nucleotide excision repair (NER) in a fragment ∼25 to 30 nucleotides long. In humans, a deficiency in NER causes xeroderma pigmentosum (XP), characterized by extreme sensitivity to sunlight and a high incidence of skin cancers. Abasic (AP) sites are formed in DNA as a result of spontaneous base loss and from the action of DNA glycosylases involved in base excision repair. In Saccharomyces cerevisiae, AP sites are removed via the action of two class II AP endonucleases, Apn1 and Apn2. Here, we provide evidence for the involvement of NER in the removal of AP sites and show that NER competes with Apn1 and Apn2 in this repair process. Inactivation of NER in the apn1Δ orapn1Δ apn2Δ strain enhances sensitivity to the monofunctional alkylating agent methyl methanesulfonate and leads to further impairment in the cellular ability to remove AP sites. A deficiency in the repair of AP sites may contribute to the internal cancers and progressive neurodegeneration that occur in XP patients.

scholarly journals Origin of Endogenous DNA Abasic Sites in Saccharomyces cerevisiae

2003 ◽  
Vol 23 (22) ◽  
pp. 8386-8394 ◽  
Author(s):  
Marie Guillet ◽  
Serge Boiteux
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Delay ◽  
Dna Glycosylases ◽  
Triple Mutant ◽  
Gene Encoding ◽  
Cytosine Deamination ◽  
Abasic Sites ◽  
Single Strand Breaks ◽  
Genes Encoding ◽  
Ap Sites

ABSTRACT Abasic (AP) sites are among the most frequent endogenous lesions in DNA and present a strong block to replication. In Saccharomyces cerevisiae, an apn1 apn2 rad1 triple mutant is inviable because of its incapacity to repair AP sites and related 3′-blocked single-strand breaks (M. Guillet and S. Boiteux, EMBO J. 21:2833, 2002). Here, we investigated the origin of endogenous AP sites in yeast. Our results show that the deletion of the UNG1 gene encoding the uracil DNA glycosylase suppresses the lethality of the apn1 apn2 rad1 mutant. In contrast, inactivation of the MAG1, OGG1, or NTG1 and NTG2 genes encoding DNA glycosylases involved in the repair of alkylation or oxidation damages does not suppress lethality. Although viable, the apn1 apn2 rad1 ung1 mutant presents growth delay due to a G2/M checkpoint. These results point to uracil as a critical source of the formation of endogenous AP sites in DNA. Uracil can arise in DNA by cytosine deamination or by the incorporation of dUMP during replication. Here, we show that the overexpression of the DUT1 gene encoding the dUTP pyrophosphatase (Dut1) suppresses the lethality of the apn1 apn2 rad1 mutant. Therefore, this result points to the dUTP pool as an important source of the formation of endogenous AP sites in eukaryotes.

Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1

1984 ◽  
Vol 4 (2) ◽  
pp. 324-328
Author(s):  
C Dinsart ◽  
J J Cornelis ◽  
B Klein ◽  
A J van der Eb ◽  
J Rommelaere
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Virus ◽  
Double Stranded Dna ◽  
Mutator Activity ◽  
Calf Thymus ◽  
Circular Dnas ◽  
Uv Lesions ◽  
Damaged Dna

Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate.

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