DNA repair glycosylase hNEIL1 triages damaged bases via competing interaction modes

AbstractDNA glycosylases must distinguish the sparse damaged sites from the vast expanse of normal DNA bases. However, our understanding of the nature of nucleobase interrogation is still limited. Here, we show that hNEIL1 (human endonuclease VIII-like 1) captures base lesions via two competing states of interaction: an activated state that commits catalysis and base excision repair, and a quarantine state that temporarily separates and protects the flipped base via auto-inhibition. The relative dominance of the two states depends on key residues of hNEIL1 and chemical properties (e.g. aromaticity and hydrophilicity) of flipped bases. Such a DNA repair mechanism allows hNEIL1 to recognize a broad spectrum of DNA damage while keeps potential gratuitous repair in check. We further reveal the molecular basis of hNEIL1 activity regulation mediated by post-transcriptional modifications and provide an example of how exquisite structural dynamics serves for orchestrated enzyme functions.

Download Full-text

Formation of cytosine glycol and 5,6-dihydroxycytosine in deoxyribonucleic acid on treatment with osmium tetroxide

Biochemical Journal ◽

10.1042/bj2350531 ◽

1986 ◽

Vol 235 (2) ◽

pp. 531-536 ◽

Cited By ~ 76

Author(s):

M Dizdaroglu ◽

E Holwitt ◽

M P Hagan ◽

W F Blakely

Keyword(s):

Dna Repair ◽

Formic Acid ◽

Excision Repair ◽

Dna Lesions ◽

Gas Chromatography Mass Spectrometry ◽

Dna Glycosylases ◽

Thymine Glycol ◽

Repair Enzymes ◽

Base Excision

OsO4 selectively forms thymine glycol lesions in DNA. In the past, OsO4-treated DNA has been used as a substrate in studies of DNA repair utilizing base-excision repair enzymes such as DNA glycosylases. There is, however, no information available on the chemical identity of other OsO4-induced base lesions in DNA. A complete knowledge of such DNA lesions may be of importance for repair studies. Using a methodology developed recently for characterization of oxidative base damage in DNA, we provide evidence for the formation of cytosine glycol and 5,6-dihydroxycytosine moieties, in addition to thymine glycol, in DNA on treatment with OsO4. For this purpose, samples of OsO4-treated DNA were hydrolysed with formic acid, then trimethylsilylated and analysed by capillary gas chromatography-mass spectrometry. In addition to thymine glycol, 5-hydroxyuracil (isobarbituric acid), 5-hydroxycytosine and 5,6-dihydroxyuracil (isodialuric acid or dialuric acid) were identified in OsO4-treated DNA. It is suggested that 5-hydroxyuracil was formed by formic acid-induced deamination and dehydration of cytosine glycol, which was the actual oxidation product of the cytosine moiety in DNA. 5-Hydroxycytosine obviously resulted from dehydration of cytosine glycol, and 5,6-dihydroxyuracil from deamination of 5,6-dihydroxycytosine. This scheme was supported by the presence of 5-hydroxyuracil, uracil glycol and 5,6-dihydroxyuracil in OsO4-treated cytosine. Treatment of OsO4-treated cytosine with formic acid caused the complete conversion of uracil glycol into 5-hydroxyuracil. The implications of these findings relative to studies of DNA repair are discussed.

Download Full-text

The Base Excision Repair Pathway in the Nematode Caenorhabditis elegans

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.598860 ◽

2020 ◽

Vol 8 ◽

Author(s):

Noha Elsakrmy ◽

Qiu-Mei Zhang-Akiyama ◽

Dindial Ramotar

Keyword(s):

Dna Repair ◽

Caenorhabditis Elegans ◽

Dna Polymerase ◽

Base Excision Repair ◽

Excision Repair ◽

Dna Glycosylases ◽

Repair Synthesis ◽

C Elegans ◽

Base Excision ◽

Dna Repair Synthesis

Exogenous and endogenous damage to the DNA is inevitable. Several DNA repair pathways including base excision, nucleotide excision, mismatch, homologous and non-homologous recombinations are conserved across all organisms to faithfully maintain the integrity of the genome. The base excision repair (BER) pathway functions to repair single-base DNA lesions and during the process creates the premutagenic apurinic/apyrimidinic (AP) sites. In this review, we discuss the components of the BER pathway in the nematode Caenorhabditis elegans and delineate the different phenotypes caused by the deletion or the knockdown of the respective DNA repair gene, as well as the implications. To date, two DNA glycosylases have been identified in C. elegans, the monofunctional uracil DNA glycosylase-1 (UNG-1) and the bifunctional endonuclease III-1 (NTH-1) with associated AP lyase activity. In addition, the animal possesses two AP endonucleases belonging to the exonuclease-3 and endonuclease IV families and in C. elegans these enzymes are called EXO-3 and APN-1, respectively. In mammalian cells, the DNA polymerase, Pol beta, that is required to reinsert the correct bases for DNA repair synthesis is not found in the genome of C. elegans and the evidence indicates that this role could be substituted by DNA polymerase theta (POLQ), which is known to perform a function in the microhomology-mediated end-joining pathway in human cells. The phenotypes observed by the C. elegans mutant strains of the BER pathway raised many challenging questions including the possibility that the DNA glycosylases may have broader functional roles, as discuss in this review.

Download Full-text

Modulation of the Apurinic/Apyrimidinic Endonuclease Activity of Human APE1 and of Its Natural Polymorphic Variants by Base Excision Repair Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms21197147 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7147

Author(s):

Olga A. Kladova ◽

Irina V. Alekseeva ◽

Murat Saparbaev ◽

Olga S. Fedorova ◽

Nikita A. Kuznetsov

Keyword(s):

Dna Repair ◽

Binding Site ◽

Base Excision Repair ◽

Protein Interactions ◽

Excision Repair ◽

Dna Glycosylases ◽

Protein Protein Interactions ◽

Dna Binding Site ◽

Polymorphic Variants ◽

Base Excision

Human apurinic/apyrimidinic endonuclease 1 (APE1) is known to be a critical player of the base excision repair (BER) pathway. In general, BER involves consecutive actions of DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. It is known that these proteins interact with APE1 either at upstream or downstream steps of BER. Therefore, we may propose that even a minor disturbance of protein–protein interactions on the DNA template reduces coordination and repair efficiency. Here, the ability of various human DNA repair enzymes (such as DNA glycosylases OGG1, UNG2, and AAG; DNA polymerase Polβ; or accessory proteins XRCC1 and PCNA) to influence the activity of wild-type (WT) APE1 and its seven natural polymorphic variants (R221C, N222H, R237A, G241R, M270T, R274Q, and P311S) was tested. Förster resonance energy transfer–based kinetic analysis of abasic site cleavage in a model DNA substrate was conducted to detect the effects of interacting proteins on the activity of WT APE1 and its single-nucleotide polymorphism (SNP) variants. The results revealed that WT APE1 activity was stimulated by almost all tested DNA repair proteins. For the SNP variants, the matters were more complicated. Analysis of two SNP variants, R237A and G241R, suggested that a positive charge in this area of the APE1 surface impairs the protein–protein interactions. In contrast, variant R221C (where the affected residue is located near the DNA-binding site) showed permanently lower activation relative to WT APE1, whereas neighboring SNP N222H did not cause a noticeable difference as compared to WT APE1. Buried substitution P311S had an inconsistent effect, whereas each substitution at the DNA-binding site, M270T and R274Q, resulted in the lowest stimulation by BER proteins. Protein–protein molecular docking was performed between repair proteins to identify amino acid residues involved in their interactions. The data uncovered differences in the effects of BER proteins on APE1, indicating an important role of protein–protein interactions in the coordination of the repair pathway.

Download Full-text

Computational studies of DNA repair: Insights into the function of monofunctional DNA glycosylases in the base excision repair pathway

Wiley Interdisciplinary Reviews Computational Molecular Science ◽

10.1002/wcms.1471 ◽

2020 ◽

Vol 10 (5) ◽

Author(s):

Rajwinder Kaur ◽

Dylan J. Nikkel ◽

Stacey D. Wetmore

Keyword(s):

Dna Repair ◽

Base Excision Repair ◽

Excision Repair ◽

Computational Studies ◽

Dna Glycosylases ◽

Repair Pathway ◽

Base Excision Repair Pathway ◽

Base Excision

Download Full-text

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

Acta Biochimica Polonica ◽

10.18388/abp.2005_3503 ◽

2005 ◽

Vol 52 (1) ◽

pp. 167-178 ◽

Cited By ~ 13

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.

Download Full-text

Effect of DNA Glycosylases OGG1 and Neil1 on Oxidized G-Rich Motif in the KRAS Promoter

International Journal of Molecular Sciences ◽

10.3390/ijms22031137 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1137

Author(s):

Annalisa Ferino ◽

Luigi E. Xodo

Keyword(s):

Oxidative Stress ◽

Base Excision Repair ◽

Excision Repair ◽

Start Site ◽

Dna Glycosylases ◽

Repair Pathway ◽

Transcription Start ◽

G Quadruplex ◽

Base Excision ◽

Regulatory Functions

The promoter of the Kirsten ras (KRAS) proto-oncogene contains, upstream of the transcription start site, a quadruplex-forming motif called 32R with regulatory functions. As guanine under oxidative stress can be oxidized to 8-oxoguanine (8OG), we investigated the capacity of glycosylases 8-oxoguanine glycosylase (OGG1) and endonuclease VIII-like 1 (Neil1) to excise 8OG from 32R, either in duplex or G-quadruplex (G4) conformation. We found that OGG1 efficiently excised 8OG from oxidized 32R in duplex but not in G4 conformation. By contrast, glycosylase Neil1 showed more activity on the G4 than the duplex conformation. We also found that the excising activity of Neil1 on folded 32R depended on G4 topology. Our data suggest that Neil1, besides being involved in base excision repair pathway (BER), could play a role on KRAS transcription.

Download Full-text

An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.68 ◽

1994 ◽

Vol 14 (1) ◽

pp. 68-76 ◽

Cited By ~ 321

Author(s):

K W Caldecott ◽

C K McKeown ◽

J D Tucker ◽

S Ljungquist ◽

L H Thompson

Keyword(s):

Dna Repair ◽

Affinity Chromatography ◽

Mammalian Cells ◽

Excision Repair ◽

Affinity Purification ◽

Alkylating Agents ◽

Chinese Hamster ◽

Dna Ligase ◽

Base Excision ◽

Dna Ligase Iii

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

Download Full-text

Incorporation of 5’,8-cyclo-2’deoxyadenosines by DNA repair polymerases via base excision repair

DNA Repair ◽

10.1016/j.dnarep.2021.103258 ◽

2021 ◽

pp. 103258

Author(s):

Pawlos S. Tsegay ◽

Daniela Hernandez ◽

Christopher Brache ◽

Chryssostomos Chatgilialoglu ◽

Marios G. Krokidis ◽

...

Keyword(s):

Dna Repair ◽

Base Excision Repair ◽

Excision Repair ◽

Base Excision

Download Full-text

Molecular Mechanisms of the Whole DNA Repair System: A Comparison of Bacterial and Eukaryotic Systems

Journal of Nucleic Acids ◽

10.4061/2010/179594 ◽

2010 ◽

Vol 2010 ◽

pp. 1-32 ◽

Cited By ~ 45

Author(s):

Rihito Morita ◽

Shuhei Nakane ◽

Atsuhiro Shimada ◽

Masao Inoue ◽

Hitoshi Iino ◽

...

Keyword(s):

Dna Repair ◽

Molecular Mechanisms ◽

Excision Repair ◽

Thermus Thermophilus ◽

Repair System ◽

System A ◽

Recombination Repair ◽

Repair Mechanisms ◽

Repair Pathways ◽

Base Excision

DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly inThermus thermophilusHB8.

Download Full-text