scholarly journals Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis

2018 ◽  
Vol 115 (5) ◽  
pp. E916-E924 ◽  
Author(s):  
Casimiro Barbado ◽  
Dolores Córdoba-Cañero ◽  
Rafael R. Ariza ◽  
Teresa Roldán-Arjona
Keyword(s):  
Excision Repair ◽  
Biochemical Properties ◽  
Abasic Site ◽  
Single Nucleotide ◽  
Ap Endonuclease ◽  
Abasic Sites ◽  
Lyase Activity ◽  
Ap Sites ◽  
Base Excision ◽  
Ap Lyase

Abasic (apurinic/apyrimidinic, AP) sites in DNA arise from spontaneous base loss or by enzymatic removal during base excision repair. It is commonly accepted that both classes of AP site have analogous biochemical properties and are equivalent substrates for AP endonucleases and AP lyases, although the relative roles of these two types of enzymes are not well understood. We provide here genetic and biochemical evidence that, in Arabidopsis, AP sites generated by spontaneous loss of N7-methylguanine (N7-meG) are exclusively repaired through an AP endonuclease-independent pathway initiated by FPG, a bifunctional DNA glycosylase with AP lyase activity. Abasic site incision catalyzed by FPG generates a single-nucleotide gap with a 3′-phosphate terminus that is processed by the DNA 3′-phosphatase ZDP before repair is completed. We further show that the major AP endonuclease in Arabidopsis (ARP) incises AP sites generated by enzymatic N7-meG excision but, unexpectedly, not those resulting from spontaneous N7-meG loss. These findings, which reveal previously undetected differences between products of enzymatic and nonenzymatic base release, may shed light on the evolution and biological roles of AP endonucleases and AP lyases.

scholarly journals A non-canonical role for the DNA glycosylase NEIL3 in suppressing APE1 endonuclease-mediated ssDNA damage

2020 ◽  
Vol 295 (41) ◽  
pp. 14222-14235 ◽  
Author(s):  
Anh Ha ◽  
Yunfeng Lin ◽  
Shan Yan
Keyword(s):  
Excision Repair ◽  
Dna Glycosylase ◽  
Genome Integrity ◽  
Ap Endonuclease ◽  
C Terminus ◽  
Egg Extracts ◽  
Lyase Activity ◽  
Repair Pathways ◽  
Base Excision ◽  
Ap Lyase

The DNA glycosylase NEIL3 has been implicated in DNA repair pathways including the base excision repair and the interstrand cross-link repair pathways via its DNA glycosylase and/or AP lyase activity, which are considered canonical roles of NEIL3 in genome integrity. Compared with the other DNA glycosylases NEIL1 and NEIL2, Xenopus laevis NEIL3 C terminus has two highly conserved zinc finger motifs containing GRXF residues (designated as Zf-GRF). It has been demonstrated that the minor AP endonuclease APE2 contains only one Zf-GRF motif mediating interaction with single-strand DNA (ssDNA), whereas the major AP endonuclease APE1 does not. It appears that the two NEIL3 Zf-GRF motifs (designated as Zf-GRF repeat) are dispensable for its DNA glycosylase and AP lyase activity; however, the potential function of the NEIL3 Zf-GRF repeat in genome integrity remains unknown. Here, we demonstrate evidence that the NEIL3 Zf-GRF repeat was associated with a higher affinity for shorter ssDNA than one single Zf-GRF motif. Notably, our protein–protein interaction assays show that the NEIL3 Zf-GRF repeat but not one Zf-GRF motif interacted with APE1 but not APE2. We further reveal that APE1 endonuclease activity on ssDNA but not on dsDNA is compromised by a NEIL3 Zf-GRF repeat, whereas one Zf-GRF motif within NEIL3 is not sufficient to prevent such activity of APE1. In addition, COMET assays show that excess NEIL3 Zf-GRF repeat reduces DNA damage in oxidative stress in Xenopus egg extracts. Together, our results suggest a noncanonical role of NEIL3 in genome integrity via its distinct Zf-GRF repeat in suppressing APE1 endonuclease-mediated ssDNA breakage.

scholarly journals Complementary Functions of Plant AP Endonucleases and AP Lyases during DNA Repair of Abasic Sites Arising from C:G Base Pairs

2021 ◽  
Vol 22 (16) ◽  
pp. 8763
Author(s):  
Marina Jordano-Raya ◽  
Cristina Beltrán-Melero ◽  
M. Dolores Moreno-Recio ◽  
M. Isabel Martínez-Macías ◽  
Rafael R. Ariza ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Lesions ◽  
Abasic Site ◽  
Base Loss ◽  
Base Pairs ◽  
Ap Endonuclease ◽  
Abasic Sites ◽  
Ap Sites ◽  
Ap Lyase ◽  
Ap Site

Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from spontaneous base loss and excision of damaged bases. They may be processed either by AP endonucleases or AP lyases, but the relative roles of these two classes of enzymes are not well understood. We hypothesized that endonucleases and lyases may be differentially influenced by the sequence surrounding the AP site and/or the identity of the orphan base. To test this idea, we analysed the activity of plant and human AP endonucleases and AP lyases on DNA substrates containing an abasic site opposite either G or C in different sequence contexts. AP sites opposite G are common intermediates during the repair of deaminated cytosines, whereas AP sites opposite C frequently arise from oxidized guanines. We found that the major Arabidopsis AP endonuclease (ARP) exhibited a higher efficiency on AP sites opposite G. In contrast, the main plant AP lyase (FPG) showed a greater preference for AP sites opposite C. The major human AP endonuclease (APE1) preferred G as the orphan base, but only in some sequence contexts. We propose that plant AP endonucleases and AP lyases play complementary DNA repair functions on abasic sites arising at C:G pairs, neutralizing the potential mutagenic consequences of C deamination and G oxidation, respectively.

scholarly journals Inhibition of DNA repair glycosylases by base analogs and tryptophan pyrolysate, Trp-P-1.

2005 ◽  
Vol 52 (1) ◽  
pp. 167-178 ◽  
Author(s):  
Elzbieta Speina ◽  
Jarosław M Cieśla ◽  
Maria-Anna Graziewicz ◽  
Jacques Laval ◽  
Zygmunt Kazimierczuk ◽  
...  
Keyword(s):  
Dna Repair ◽  
Excision Repair ◽  
Dna Structure ◽  
Dna Glycosylases ◽  
Ap Endonuclease ◽  
Abasic Sites ◽  
Repair Enzymes ◽  
Lyase Activity ◽  
Base Analog ◽  
Base Excision

DNA base analogs, 2,4,5,6-substituted pyrimidines and 2,6-substituted purines were tested as potential inhibitors of E. coli Fpg protein (formamidopyrimidine -DNA glycosylase). Three of the seventeen compounds tested revealed inhibitory properties. 2-Thioxanthine was the most efficient, inhibiting 50% of 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeG) excision activity at 17.1 microM concentration. The measured K(i) was 4.44 +/- 0.15 microM. Inhibition was observed only when the Fpg protein was first challenged to its substrate followed by the addition of the base analog, suggesting uncompetitive (catalytic) inhibition. For two other compounds, 2-thio- or 2-oxo-4,5,6-substituted pyrimidines, IC(50) was only 343.3 +/- 58.6 and 350 +/- 24.4 microM, respectively. No change of the Fpg glycosylase activity was detected in the presence of Fapy-7MeG, up to 5 microM. We also investigated the effect of DNA structure modified by tryptophan pyrolysate (Trp-P-1) on the activity of base excision repair enzymes: Escherichia coli and human DNA glycosylases of oxidized (Fpg, Nth) and alkylated bases (TagA, AlkA, and ANPG), and for bacterial AP endonuclease (Xth protein). Trp-P-1, which changes the secondary DNA structure into non-B, non-Z most efficiently inhibited excision of alkylated bases by the AlkA glycosylase (IC(50) = 1 microM). The ANPG, TagA, and Fpg proteins were also inhibited although to a lesser extent (IC(50) = 76.5 microM, 96 microM, and 187.5 microM, respectively). Trp-P-1 also inhibited incision of DNA at abasic sites by the beta-lyase activity of the Fpg and Nth proteins, and to a lesser extent by the Xth AP endonuclease. Thus, DNA conformation is critical for excision of damaged bases and incision of abasic sites by DNA repair enzymes.

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.

Chemical and biochemical properties of abasic site adducts and 6-thioguanine DNA cross-links in DNA

2017 ◽  
Author(s):  
◽  
Calvin D. Lewis
Keyword(s):  
Excision Repair ◽  
Biochemical Properties ◽  
Macromolecular Complex ◽  
Abasic Site ◽  
Cross Link ◽  
Dna Lesion ◽  
Phosphate Backbone ◽  
Cross Links ◽  
Ap Sites ◽  
Cellular Dna

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] DNA is a macromolecular complex, composed of the nucleotides adenine, thymine, guanine and cytosine interconnected by a phosphate backbone, that contains the genetic code for living organisms and viruses. Spontaneous and enzymatic hydrolysis of the glycosidic bonds that hold the coding nucleobases to the 2-deoxyribose-phosphate backbone of DNA results in the production of abasic (Ap) sites. These lesions are abundant in cellular DNA, and cellular Ap-containing DNA is damaging and may lead to cellular destruction if left unrepaired. Thus, efficient cellular DNA repair mechanisms that repair Ap sites have evolved in DNA containing organisms. The studies in this report examine the interaction between small molecules or naturally occurring DNA residues with Ap sites in duplex DNA. Experiments provide evidence that hydralazine binds to and forms a stable DNA lesion in single- and double-stranded DNA. Also, the hydralazine-DNA lesion is found to be a poor substrate for mammalian base excision repair enzymes such as Ap endonuclease and 8-oxoguanine DNA glycosylase. In addition, these studies provide preliminary evidence that hydralazine may potentiate the cytotoxicity of temozolomide in U87 cells. The investigation of the formation of cross-links between canonical DNA residues deoxyadenosine (dA) and deoxyguanosine (dG) with Ap sites is also explored. These experiments suggest that sequence effects contribute majorly to the cross-link yield in both dA- and dG-Ap site cross-links, especially when comparing central versus terminal cross-link locations. Here, this manuscript provides novel studies involving the interaction between DNA analog 6-thioguanine and opposing DNA bases in duplex oligonucleotide DNA.

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 Efficient Repair of Abasic Sites in DNA by Mitochondrial Enzymes

1998 ◽  
Vol 18 (3) ◽  
pp. 1257-1265 ◽  
Author(s):  
Kevin G. Pinz ◽  
Daniel F. Bogenhagen
Keyword(s):  
Nuclear Dna ◽  
Dna Ligase ◽  
Mitochondrial Enzymes ◽  
Degenerative Diseases ◽  
Abasic Site ◽  
Repair Pathway ◽  
Ap Endonuclease ◽  
Abasic Sites ◽  
Dna Backbone ◽  
Base Excision

ABSTRACT Mutations in mitochondrial DNA (mtDNA) cause a variety of relatively rare human diseases and may contribute to the pathogenesis of other, more common degenerative diseases. This stimulates interest in the capacity of mitochondria to repair damage to mtDNA. Several recent studies have shown that some types of damage to mtDNA may be repaired, particularly if the lesions can be processed through a base excision mechanism that employs an abasic site as a common intermediate. In this paper, we demonstrate that a combination of enzymes purified from Xenopus laevis mitochondria efficiently repairs abasic sites in DNA. This repair pathway employs a mitochondrial class II apurinic/apyrimidinic (AP) endonuclease to cleave the DNA backbone on the 5′ side of an abasic site. A deoxyribophosphodiesterase acts to remove the 5′ sugar-phosphate residue left by AP endonuclease. mtDNA polymerase γ fills the resulting 1-nucleotide gap. The remaining nick is sealed by an mtDNA ligase. We report the first extensive purification of mtDNA ligase as a 100-kDa enzyme that functions with an enzyme-adenylate intermediate and is capable of ligating oligo(dT) strands annealed to poly(rA). These properties together with preliminary immunological evidence suggest that mtDNA may be related to nuclear DNA ligase III.

scholarly journals The use of thioglycollate to demonstrate DNA AP (apurinic/apyrimidinic-site) lyase activities. Biological consequences of thiol addition to the 5′ product of a β-elimination reaction at an AP site in DNA

1991 ◽  
Vol 273 (3) ◽  
pp. 777-782 ◽  
Author(s):  
S Bricteux-Grégoire ◽  
W G Verly
Keyword(s):  
Escherichia Coli ◽  
Elimination Reaction ◽  
Ap Endonuclease ◽  
Lyase Activity ◽  
Ap Sites ◽  
Ap Lyase ◽  
Ap Site

Thioglycollate reacts with the 5′ product of AP lyase activity on apurinic/apyrimidinic (AP) sites in DNA. The 3′-terminal thioglycollate-unsaturated sugar 5-phosphate adduct can be released by the use of Escherichia coli endonuclease IV or endonuclease VI, and identified by DEAE-Sephadex chromatography. In contrast, the mammalian AP endonuclease is unable to excise a 3′-terminal thiol-unsaturated sugar adduct; this lesion, which must sometimes occur in vivo, might be irreparable and have pathological consequences.

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