Effects of 5′,8′-Cyclo-2′-Deoxypurines on the Base ExcisionRepair of Clustered DNA Lesions in Nuclear Extracts of the XPC Cell Line

Clustered DNA lesions (CDL) containing 5′,8-cyclo-2′-deoxypurines (cdPus) are an example of extensive abnormalities occurring in the DNA helix and may impede cellular repair processes. The changes in the efficiency of nuclear base excision repair (BER) were investigated using (a) two cell lines, one of the normal skin fibroblasts as a reference (BJ) and the second from Xeroderma pigmentosum patients’ skin (XPC), and (b) synthetic oligonucleotides with single- and double-stranded CDL (containing 5′,8-cyclo-2′-deoxyadenosine (cdA) and the abasic (AP) site at various distances between lesions). The nuclear BER has been observed and the effect of both cdA isomers (5′R and 5′S) presence in the DNA was tested. CdPus affected the repair of the second lesion within the CDL. The BER system more efficiently processed damage in the vicinity of the ScdA isomer and changes located in the 3′-end direction for dsCDL and in the 5′-end direction for ssCDL. The presented study is the very first investigation of the repair processes of the CDL containing cdPu considering cells derived from a Xeroderma pigmentosum patient.

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When UDG and hAPE1 Meet Cyclopurines. How (5′R) and (5′S) 5′,8-Cyclo-2′-deoxyadenosine and 5′,8-Cyclo-2′-deoxyguanosine Affect UDG and hAPE1 Activity?

Molecules ◽

10.3390/molecules26175177 ◽

2021 ◽

Vol 26 (17) ◽

pp. 5177

Author(s):

Michał Szewczuk ◽

Karolina Boguszewska ◽

Julia Kaźmierczak-Barańska ◽

Bolesław T. Karwowski

Keyword(s):

Base Excision Repair ◽

Excision Repair ◽

Dna Lesions ◽

Repair System ◽

Cellular Mechanisms ◽

Uracil Dna Glycosylase ◽

Base Excision ◽

Base Excision Repair System ◽

Synthetic Oligonucleotides ◽

Ap Site

Ionizing radiation is a factor that seriously damages cellular mechanisms/macromolecules, e.g., by inducing damage in the human genome, such as 5′,8-cyclo-2′-deoxypurines (cdPus). CdPus may become a component of clustered DNA lesions (CDL), which are notably unfavorable for the base excision repair system (BER). In this study, the influence of 5′S and 5′R diastereomers of 5′,8-cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) on the uracil-DNA glycosylase (UDG) and human AP site endonuclease 1 (hAPE1) activity has been taken under consideration. Synthetic oligonucleotides containing 2′-deoxyuridine (dU) and cdPu were used as a model of single-stranded CDL. The activity of the UDG and hAPE1 enzymes decreased in the presence of RcdG compared to ScdG. Contrary to the above, ScdA reduced enzyme activity more than RcdA. The presented results show the influence of cdPus lesions located within CDL on the activity of the initial stages of BER dependently on their position toward dU. Numerous studies have shown the biological importance of cdPus (e.g., as a risk of carcinogenesis). Due to that, it is important to understand how to recognize and eliminate this type of DNA damage from the genome.

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The Influence of (5′R)- and (5′S)-5′,8-Cyclo-2′-Deoxyadenosine on UDG and hAPE1 Activity. Tandem Lesions are the Base Excision Repair System’s Nightmare

Cells ◽

10.3390/cells8111303 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1303 ◽

Cited By ~ 2

Author(s):

Karwowski

Keyword(s):

Dna Damage ◽

Base Excision Repair ◽

Excision Repair ◽

Repair Process ◽

Dna Lesions ◽

Repair System ◽

Base Excision ◽

Base Excision Repair System ◽

Tandem Lesions ◽

Ap Site

DNA lesions are formed continuously in each living cell as a result of environmental factors, ionisation radiation, metabolic processes, etc. Most lesions are removed from the genome by the base excision repair system (BER). The activation of the BER protein cascade starts with DNA damage recognition by glycosylases. Uracil-DNA glycosylase (UDG) is one of the most evolutionary preserved glycosylases which remove the frequently occurring 2′-deoxyuridine from single (ss) and double-stranded (ds) oligonucleotides. Conversely, the unique tandem lesions (5′R)- and (5′S)-5′,8-cyclo-2′-deoxyadenosine (cdA) are not suitable substrates for BER machinery and are released from the genome by the nucleotide excision repair (NER) system. However, the cyclopurines appearing in a clustered DNA damage structure can influence the BER process of other lesions like dU. In this article, UDG inhibition by 5′S- and 5′R-cdA is shown and discussed in an experimental and theoretical manner. This phenomenon was observed when a tandem lesion appears in single or double-stranded oligonucleotides next to dU, on its 3′-end side. The cdA shift to the 5′-end side of dU in ss-DNA stops this effect in both cdA diastereomers. Surprisingly, in the case of ds-DNA, 5′S-cdA completely blocks uracil excision by UDG. Conversely, 5′R-cdA allows glycosylase for uracil removal, but the subsequently formed apurinic/apyrimidinic (AP) site is not suitable for human AP-site endonuclease 1 (hAPE1) activity. In conclusion, the appearance of the discussed tandem lesion in the structure of single or double-stranded DNA can stop the entire base repair process at its beginning, which due to UDG and hAPE1 inhibition can lead to mutagenesis. On the other hand, the presented results can cast some light on the UDG or hAPE1 inhibitors being used as a potential treatment.

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The Influence of 5′,8-Cyclo-2′-deoxypurines on the Mitochondrial Repair of Clustered DNA Damage in Xrs5 Cells: The Preliminary Study

Molecules ◽

10.3390/molecules26227042 ◽

2021 ◽

Vol 26 (22) ◽

pp. 7042

Author(s):

Karolina Boguszewska ◽

Julia Kaźmierczak-Barańska ◽

Bolesław T. Karwowski

Keyword(s):

Excision Repair ◽

Dna Lesions ◽

Base Pairs ◽

Apurinic Site ◽

Ap Sites ◽

Base Excision ◽

First Time ◽

Ap Site ◽

Clustered Dna Damage ◽

Single Lesion

The 5′,8-cyclo-2′-deoxypurines (cdPus) affect the DNA structure. When these bulky structures are a part of clustered DNA lesions (CDL), they affect the repair of the other lesions within the cluster. Mitochondria are crucial for cell survival and have their own genome, hence, are highly interesting in the context of CDL repair. However, no studies are exploring this topic. Here, the initial stages of mitochondrial base excision repair (mtBER) were considered—the strand incision and elongation. The repair of a single lesion (apurinic site (AP site)) accompanying the cdPu within the double-stranded CDL has been investigated for the first time. The type of cdPu, its diastereomeric form, and the interlesion distance were taken into consideration. For these studies, the established experimental model of short oligonucleotides (containing AP sites located ≤7 base pairs to the cdPu in both directions) and mitochondrial extracts of the xrs5 cells were used. The obtained results have shown that the presence of cdPus influenced the processing of an AP site within the CDL. Levels of strand incision and elongation were higher for oligos containing RcdA and ScdG than for those with ScdA and RcdG. Investigated stages of mtBER were more efficient for DNA containing AP sites located on 5′-end side of cdPu than on its 3′-end side. In conclusion, the presence of cdPus in mtDNA structure may affect mtBER (processing the second mutagenic lesion within the CDL). As impaired repair processes may lead to serious biological consequences, further studies concerning the mitochondrial repair of CDL are highly demanded.

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How (5′S) and (5′R) 5′,8-Cyclo-2′-Deoxypurines Affect Base Excision Repair of Clustered DNA Damage in Nuclear Extracts of xrs5 Cells? A Biochemical Study

Cells ◽

10.3390/cells10040725 ◽

2021 ◽

Vol 10 (4) ◽

pp. 725

Author(s):

Karolina Boguszewska ◽

Michał Szewczuk ◽

Julia Kaźmierczak-Barańska ◽

Bolesław T. Karwowski

Keyword(s):

Base Excision Repair ◽

Excision Repair ◽

Genetic Material ◽

Dna Lesions ◽

The Other ◽

Base Pairs ◽

Ap Sites ◽

Base Excision ◽

The Impact ◽

Ap Site

The clustered DNA lesions (CDLs) are a characteristic feature of ionizing radiation’s impact on the human genetic material. CDLs impair the efficiency of cellular repair machinery, especially base excision repair (BER). When CDLs contain a lesion repaired by BER (e.g., apurinic/apyrimidinic (AP) sites) and a bulkier 5′,8-cyclo-2′-deoxypurine (cdPu), which is not a substrate for BER, the repair efficiency of the first one may be affected. The cdPus’ influence on the efficiency of nuclear BER in xrs5 cells have been investigated using synthetic oligonucleotides with bi-stranded CDL (containing (5′S) 5′,8-cyclo-2′-deoxyadenosine (ScdA), (5′R) 5′,8-cyclo-2′-deoxyadenosine (RcdA), (5′S) 5′,8-cyclo-2′-deoxyguanosine (ScdG) or (5′R) 5′,8-cyclo-2′-deoxyguanosine (RcdG) in one strand and an AP site in the other strand at different interlesion distances). Here, for the first time, the impact of ScdG and RcdG was experimentally tested in the context of nuclear BER. This study shows that the presence of RcdA inhibits BER more than ScdA; however, ScdG decreases repair level more than RcdG. Moreover, AP sites located ≤10 base pairs to the cdPu on its 5′-end side were repaired less efficiently than AP sites located ≤10 base pairs on the 3′-end side of cdPu. The strand with an AP site placed opposite cdPu or one base in the 5′-end direction was not reconstituted for cdA nor cdG. CdPus affect the repair of the other lesion within the CDL. It may translate to a prolonged lifetime of unrepaired lesions leading to mutations and impaired cellular processes. Therefore, future research should focus on exploring this subject in more detail.

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

Molecular and Cellular Biology ◽

10.1128/mcb.00308-10 ◽

2010 ◽

Vol 30 (13) ◽

pp. 3206-3215 ◽

Cited By ~ 45

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.

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Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

Frontiers in Microbiology ◽

10.3389/fmicb.2021.608839 ◽

2021 ◽

Vol 12 ◽

Author(s):

Almaz Nigatu Tesfahun ◽

Marina Alexeeva ◽

Miglė Tomkuvienė ◽

Aysha Arshad ◽

Prashanna Guragain ◽

...

Keyword(s):

Escherichia Coli ◽

Excision Repair ◽

Dna Lesions ◽

Base Pairs ◽

E Coli ◽

Thymine Glycol ◽

Base Excision ◽

The Cost ◽

Primary Substrate

DNA polymerase III mis-insertion may, where not corrected by its 3′→ 5′ exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The kcat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo8G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The KM values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N4- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

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Endogenous oxidized DNA bases and APE1 regulate the formation of G-quadruplex structures in the genome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1912355117 ◽

2020 ◽

Vol 117 (21) ◽

pp. 11409-11420 ◽

Cited By ~ 4

Author(s):

Shrabasti Roychoudhury ◽

Suravi Pramanik ◽

Hannah L. Harris ◽

Mason Tarpley ◽

Aniruddha Sarkar ◽

...

Keyword(s):

Excision Repair ◽

Factor Loading ◽

Telomere Maintenance ◽

Epigenetic Mechanism ◽

Dna Bases ◽

G Quadruplex ◽

Base Excision ◽

Ap Site ◽

In Cells

Formation of G-quadruplex (G4) DNA structures in key regulatory regions in the genome has emerged as a secondary structure-based epigenetic mechanism for regulating multiple biological processes including transcription, replication, and telomere maintenance. G4 formation (folding), stabilization, and unfolding must be regulated to coordinate G4-mediated biological functions; however, how cells regulate the spatiotemporal formation of G4 structures in the genome is largely unknown. Here, we demonstrate that endogenous oxidized guanine bases in G4 sequences and the subsequent activation of the base excision repair (BER) pathway drive the spatiotemporal formation of G4 structures in the genome. Genome-wide mapping of occurrence of Apurinic/apyrimidinic (AP) site damage, binding of BER proteins, and G4 structures revealed that oxidized base-derived AP site damage and binding of OGG1 and APE1 are predominant in G4 sequences. Loss of APE1 abrogated G4 structure formation in cells, which suggests an essential role of APE1 in regulating the formation of G4 structures in the genome. Binding of APE1 to G4 sequences promotes G4 folding, and acetylation of APE1, which enhances its residence time, stabilizes G4 structures in cells. APE1 subsequently facilitates transcription factor loading to the promoter, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels a role of endogenous oxidized DNA bases and APE1 in controlling the formation of higher-order DNA secondary structures to regulate transcription beyond its well-established role in safeguarding the genomic integrity.

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Oxygen-Dependent Accumulation of Purine DNA Lesions in Cockayne Syndrome Cells

Cells ◽

10.3390/cells9071671 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1671 ◽

Cited By ~ 1

Author(s):

Marios G. Krokidis ◽

Mariarosaria D’Errico ◽

Barbara Pascucci ◽

Eleonora Parlanti ◽

Annalisa Masi ◽

...

Keyword(s):

Dna Damage ◽

Oxidative Dna Damage ◽

Excision Repair ◽

Enzymatic Digestion ◽

Cockayne Syndrome ◽

Dna Lesions ◽

Premature Aging ◽

Neurological Features ◽

Oxygen Tensions ◽

Base Excision

Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5′,8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3–6 lesions/106 nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage.

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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.

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Databases and Bioinformatics Tools for the Study of DNA Repair

Molecular Biology International ◽

10.4061/2011/475718 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Kaja Milanowska ◽

Kristian Rother ◽

Janusz M. Bujnicki

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Excision Repair ◽

Uv Light ◽

Dna Lesions ◽

Environmental Chemicals ◽

Nonhomologous End Joining ◽

End Joining ◽

Repair Mechanisms ◽

Base Excision

DNA is continuously exposed to many different damaging agents such as environmental chemicals, UV light, ionizing radiation, and reactive cellular metabolites. DNA lesions can result in different phenotypical consequences ranging from a number of diseases, including cancer, to cellular malfunction, cell death, or aging. To counteract the deleterious effects of DNA damage, cells have developed various repair systems, including biochemical pathways responsible for the removal of single-strand lesions such as base excision repair (BER) and nucleotide excision repair (NER) or specialized polymerases temporarily taking over lesion-arrested DNA polymerases during the S phase in translesion synthesis (TLS). There are also other mechanisms of DNA repair such as homologous recombination repair (HRR), nonhomologous end-joining repair (NHEJ), or DNA damage response system (DDR). This paper reviews bioinformatics resources specialized in disseminating information about DNA repair pathways, proteins involved in repair mechanisms, damaging agents, and DNA lesions.

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