Processing of oxidatively damaged DNA dirty ends by APE1

Reactive oxygen species attack the structure of DNA, thus altering its base-pairing properties. Consequently, oxidative stress-associated DNA lesions are a major source of the mutation load that gives rise to cancer and other diseases. Base excision repair (BER) is the pathway primarily tasked with repairing DNA base damage, with apurinic/apyrimidinic endonuclease (APE1) having both APendonuclease and 3' to 5' exonuclease (exo) DNA cleavage functions. The lesion 8-oxo-7,8- dihydroguanine (8-oxoG) can enter the genome as either a product of direct damage to the DNA, or through polymerase insertion at the 3'-end of a DNA strand during replication or repair. Importantly, 3'-8-oxoG impairs the ligation step of BER and therefore must be removed by the exo activity of a surrogate enzyme to prevent double stranded breaks and cell death. In the present study, we characterize the exo activity of APE1 on 3'-8-oxoG substrates. These structures demonstrate that APE1 uses a unified mechanism for its exo activities that differs from its more canonical APendonuclease activity. In addition, through complementation of the structural data with enzyme kinetics and binding studies employing both wild-type and rationally designed APE1 mutants, we were able to identify and characterize unique protein:DNA contacts that specifically mediate 8-oxoG removal by APE1.

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Base Excision Repair Glycosylase Activity Is Impaired in a Subgroup of Acute Myeloid Leukemia Resulting in Increased Levels of Oxidative Base Lesions

Blood ◽

10.1182/blood.v124.21.860.860 ◽

2014 ◽

Vol 124 (21) ◽

pp. 860-860

Author(s):

Werner Olipitz ◽

Karin Lind ◽

Nicole Monsberger ◽

Anna Katschnig ◽

Aswin Mangerich ◽

...

Keyword(s):

Gene Expression ◽

Comet Assay ◽

Cell Lines ◽

Base Excision Repair ◽

Myeloid Leukemia ◽

Excision Repair ◽

Dna Base ◽

Base Excision ◽

Dna Strand ◽

Acute Myeloid

Abstract Base excision repair (BER) is the primary DNA repair mechanism dealing with oxidative base lesions. Oxidative DNA base lesions are the predominant type of DNA damage in mammalian cells. Deficiencies in glycosylases, the BER initiating enzymes, have been associated with increased genomic instability and increased frequencies of cancer. Here we investigated the role of oxidative BER in acute myeloid leukemia (AML). We determined oxidative BER activity in 99 primary AML blast cell samples, 34 CD34+ umbilical cord blood cell samples and 27 AML cell lines using the alkaline comet assay. Oxidative base lesion levels were determined in 10 AML cell lines using a modified version of the Comet assay with the bacterial enzymes Fpg and Endo III as well as using liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Using nuclear protein extracts in an oligonucleotide incision assay we tested the enzymatic activity of oxidative glycosylases. Finally, mutational analysis, gene expression analysis and protein expression of oxidative glycosylases was used using Sanger sequencing, real time PCR and western blot of nuclear extracts, respectively. We found DNA strand incision of oxidatively damaged bases significantly impaired in primary AML cells as compared to UCB cells (p= 0.003) suggesting a deficiency in BER glycosylases. In addition, 5/27 AML cell lines showed impaired DNA strand incision activity. We hypothesized that BER deficient cells harbor an increased number of oxidative base lesions compared to BER proficient cells. Using a modified comet assay and LC-MS/MS we were able to show that increased numbers of unrepaired oxidative base lesions were indeed present in glycosylase deficient AML cells (comet assay: p= 0.0001; mass spec: p= 0.03). We then evaluated the activity of the predominant oxidative DNA glycosylase, OGG1, and found significantly decreased DNA strand incision activity in BER deficient cells as compared to proficient cells (p= 0.002) further supporting the fact that glycosylases are impaired in BER deficient cells. Determining causes of BER deficiency preliminary experiments showed significantly decreased expression of nuclear OGG1 protein in BER deficient cells but did not reveal novel non-synonymous mutations or a difference in gene expression. Taken together we found impaired BER glycosylases in a substantial number of primary AML samples and AML cell lines resulting in increased levels of potentially mutagenic oxidative DNA base lesions Disclosures No relevant conflicts of interest to declare.

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Endonuclease IV Is the Main Base Excision Repair Enzyme Involved in DNA Damage Induced by UVA Radiation and Stannous Chloride

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/376218 ◽

2010 ◽

Vol 2010 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Ellen S. Motta ◽

Paulo Thiago Souza-Santos ◽

Tuany R. Cassiano ◽

Flávio J. S. Dantas ◽

Adriano Caldeira-de-Araujo ◽

...

Keyword(s):

Stannous Chloride ◽

Excision Repair ◽

Dna Lesions ◽

Dna Breaks ◽

Strand Breaks ◽

E Coli ◽

Dna Lesion ◽

Base Excision ◽

Dna Strand ◽

Uva Radiation

Stannous chloride (SnCl2) and UVA induce DNA lesions through ROS. The aim of this work was to study the toxicity induced by UVA preillumination, followed bySnCl2treatment.E. coliBER mutants were used to identify genes which could play a role in DNA lesion repair generated by these agents. The survival assays showed (i) Thenfomutant was the most sensitive toSnCl2; (ii) lethal synergistic effect was observed after UVA pre-illumination, plusSnCl2incubation, thenfomutant being the most sensitive; (iii) wild type andnfomutants, transformed with pBW21 plasmid (nfo+) had their survival increased following treatments. The alkaline agarose gel electrophoresis assays pointed that (i) UVA induced DNA breaks andfpgmutant was the most sensitive; (ii)SnCl2-induced DNA strand breaks were higher than those from UVA andnfomutant had the slowest repair kinetics; (iii)UVA+SnCl2promoted an increase in DNA breaks thanSnCl2and, again,nfomutant displayed the slowest repair kinetics. In summary, Nfo protectsE. colicells against damage induced bySnCl2andUVA+SnCl2.

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Rational Inhibitors of DNA Base Excision Repair (BER) Enzymes: New Tools for Elucidating the Role of the BER in Cancer Chemotherapy

10.21236/ada437740 ◽

2005 ◽

Author(s):

Daniel J. Krosky

Keyword(s):

Cancer Chemotherapy ◽

Base Excision Repair ◽

Excision Repair ◽

Dna Base Excision Repair ◽

Dna Base ◽

Base Excision

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DNA base excision repair and nucleotide excision repair proteins in malignant salivary gland tumors

Archives of Oral Biology ◽

10.1016/j.archoralbio.2020.104987 ◽

2021 ◽

Vol 121 ◽

pp. 104987

Author(s):

Fernanda Aragão Felix ◽

Leorik Pereira da Silva ◽

Maria Luiza Diniz de Sousa Lopes ◽

Ana Paula Veras Sobral ◽

Roseana de Almeida Freitas ◽

...

Keyword(s):

Salivary Gland ◽

Nucleotide Excision Repair ◽

Base Excision Repair ◽

Excision Repair ◽

Salivary Gland Tumors ◽

Dna Base Excision Repair ◽

Dna Base ◽

Nucleotide Excision ◽

Base Excision ◽

Malignant Salivary Gland Tumors

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Excision of Oxidatively Generated Guanine Lesions by Competitive DNA Repair Pathways

International Journal of Molecular Sciences ◽

10.3390/ijms22052698 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2698

Author(s):

Vladimir Shafirovich ◽

Nicholas E. Geacintov

Keyword(s):

Excision Repair ◽

Dna Lesions ◽

Dna Templates ◽

Circular Dna ◽

Damage Sensing ◽

Cell Extracts ◽

Dna Base ◽

Nucleotide Excision ◽

Repair Pathways ◽

Cellular Dna

The base and nucleotide excision repair pathways (BER and NER, respectively) are two major mechanisms that remove DNA lesions formed by the reactions of genotoxic intermediates with cellular DNA. It is generally believed that small non-bulky oxidatively generated DNA base modifications are removed by BER pathways, whereas DNA helix-distorting bulky lesions derived from the attack of chemical carcinogens or UV irradiation are repaired by the NER machinery. However, existing and growing experimental evidence indicates that oxidatively generated DNA lesions can be repaired by competitive BER and NER pathways in human cell extracts and intact human cells. Here, we focus on the interplay and competition of BER and NER pathways in excising oxidatively generated guanine lesions site-specifically positioned in plasmid DNA templates constructed by a gapped-vector technology. These experiments demonstrate a significant enhancement of the NER yields in covalently closed circular DNA plasmids (relative to the same, but linearized form of the same plasmid) harboring certain oxidatively generated guanine lesions. The interplay between the BER and NER pathways that remove oxidatively generated guanine lesions are reviewed and discussed in terms of competitive binding of the BER proteins and the DNA damage-sensing NER factor XPC-RAD23B to these lesions.

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Staring at the Naked Goddess: Unraveling the Structure and Reactivity of Artemis Endonuclease Interacting with a DNA Double Strand

Molecules ◽

10.3390/molecules26133986 ◽

2021 ◽

Vol 26 (13) ◽

pp. 3986

Author(s):

Cécilia Hognon ◽

Antonio Monari

Keyword(s):

Dna Cleavage ◽

Catalytic Mechanism ◽

Dna Lesions ◽

Cleavage Reaction ◽

Dynamic Simulations ◽

Important Target ◽

System Adaptation ◽

Dna Strands ◽

Dna Strand ◽

Dna Substrate

Artemis is an endonuclease responsible for breaking hairpin DNA strands during immune system adaptation and maturation as well as the processing of potentially toxic DNA lesions. Thus, Artemis may be an important target in the development of anticancer therapy, both for the sensitization of radiotherapy and for immunotherapy. Despite its importance, its structure has been resolved only recently, and important questions concerning the arrangement of its active center, the interaction with the DNA substrate, and the catalytic mechanism remain unanswered. In this contribution, by performing extensive molecular dynamic simulations, both classically and at the hybrid quantum mechanics/molecular mechanics level, we evidenced the stable interaction modes of Artemis with a model DNA strand. We also analyzed the catalytic cycle providing the free energy profile and key transition states for the DNA cleavage reaction.

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