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

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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Reduced Nuclease Activity of Apurinic/Apyrimidinic Endonuclease (APE1) Variants on Nucleosomes

Journal of Biological Chemistry ◽

10.1074/jbc.m115.665547 ◽

2015 ◽

Vol 290 (34) ◽

pp. 21067-21075 ◽

Cited By ~ 17

Author(s):

John M. Hinz ◽

Peng Mao ◽

Daniel R. McNeill ◽

David M. Wilson

Keyword(s):

Excision Repair ◽

Nuclease Activity ◽

Nucleosome Core Particle ◽

Nucleosome Core ◽

Efficient Processing ◽

Ap Sites ◽

Mammalian Enzyme ◽

Base Excision ◽

Dna Complex ◽

Ap Site

Non-coding apurinic/apyrimidinic (AP) sites are generated at high frequency in genomic DNA via spontaneous hydrolytic, damage-induced or enzyme-mediated base release. AP endonuclease 1 (APE1) is the predominant mammalian enzyme responsible for initiating removal of mutagenic and cytotoxic abasic lesions as part of the base excision repair (BER) pathway. We have examined here the ability of wild-type (WT) and a collection of variant/mutant APE1 proteins to cleave at an AP site within a nucleosome core particle. Our studies indicate that, in comparison to the WT protein and other variant/mutant enzymes, the incision activity of the tumor-associated variant R237C and the rare population variant G241R are uniquely hypersensitive to nucleosome complexes in the vicinity of the AP site. This defect appears to stem from an abnormal interaction of R237C and G241R with abasic DNA substrates, but is not simply due to a DNA binding defect, as the site-specific APE1 mutant Y128A, which displays markedly reduced AP-DNA complex stability, did not exhibit a similar hypersensitivity to nucleosome structures. Notably, this incision defect of R237C and G241R was observed on a pre-assembled DNA glycosylase·AP-DNA complex as well. Our results suggest that the BER enzyme, APE1, has acquired distinct surface residues that permit efficient processing of AP sites within the context of protein-DNA complexes independent of classic chromatin remodeling mechanisms.

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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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Apurinic/Apyrimidinic Endonuclease 1 and Tyrosyl-DNA Phosphodiesterase 1 Prevent Suicidal Covalent DNA-Protein Crosslink at Apurinic/Apyrimidinic Site

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.617301 ◽

2021 ◽

Vol 8 ◽

Author(s):

Natalia A. Lebedeva ◽

Nadejda I. Rechkunova ◽

Anton V. Endutkin ◽

Olga I. Lavrik

Keyword(s):

Dna Repair ◽

Excision Repair ◽

Regulatory Protein ◽

Repair Enzyme ◽

Dna Structures ◽

Ap Sites ◽

Base Excision ◽

Protein Crosslinks ◽

Covalent Complex ◽

Ap Site

Bifunctional 8-oxoguanine-DNA glycosylase (OGG1), a crucial DNA-repair enzyme, removes from DNA 8-oxo-7,8-dihydroguanine (8-oxoG) with following cleavage of the arising apurinic/apyrimidinic (AP) site. The major enzyme in eukaryotic cells that catalyzes the cleavage of AP sites is AP endonuclease 1 (APE1). Alternatively, AP sites can be cleaved by tyrosyl-DNA phosphodiesterase 1 (TDP1) to initiate APE1-independent repair, thus expanding the ability of the base excision repair (BER) process. Poly(ADP-ribose) polymerase 1 (PARP1) is a regulatory protein of DNA repair. PARP2 is also activated in response to DNA damage and can be regarded as the BER participant. Here we analyze PARP1 and PARP2 interactions with DNA intermediates of the initial stages of the BER process (8-oxoG and AP-site containing DNA) and their interplay with the proteins recognizing and processing these DNA structures focusing on OGG1. OGG1 as well as PARP1 and PARP2 form covalent complex with AP site-containing DNA without borohydride reduction. AP site incision by APE1 or TDP1 removal of protein adducts but not proteins’ PARylation prevent DNA-protein crosslinks.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22168763 ◽

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.

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Nucleotide excision repair of abasic DNA lesions

Nucleic Acids Research ◽

10.1093/nar/gkz558 ◽

2019 ◽

Vol 47 (16) ◽

pp. 8537-8547 ◽

Cited By ~ 9

Author(s):

Nataliya Kitsera ◽

Marta Rodriguez-Alvarez ◽

Steffen Emmert ◽

Thomas Carell ◽

Andriy Khobta

Keyword(s):

Nucleotide Excision Repair ◽

Excision Repair ◽

Dna Lesions ◽

Transcription Rate ◽

Repair Pathway ◽

Nucleotide Excision ◽

Repair Mechanisms ◽

Nucleotide Excision Repair Pathway ◽

Ap Sites ◽

Base Excision

AbstractApurinic/apyrimidinic (AP) sites are a class of highly mutagenic and toxic DNA lesions arising in the genome from a number of exogenous and endogenous sources. Repair of AP lesions takes place predominantly by the base excision pathway (BER). However, among chemically heterogeneous AP lesions formed in DNA, some are resistant to the endonuclease APE1 and thus refractory to BER. Here, we employed two types of reporter constructs accommodating synthetic APE1-resistant AP lesions to investigate the auxiliary repair mechanisms in human cells. By combined analyses of recovery of the transcription rate and suppression of transcriptional mutagenesis at specifically positioned AP lesions, we demonstrate that nucleotide excision repair pathway (NER) efficiently removes BER-resistant AP lesions and significantly enhances the repair of APE1-sensitive ones. Our results further indicate that core NER components XPA and XPF are equally required and that both global genome (GG-NER) and transcription coupled (TC-NER) subpathways contribute to the repair.

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

Cells ◽

10.3390/cells10113254 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3254

Author(s):

Julia Kaźmierczak-Barańska ◽

Karolina Boguszewska ◽

Michał Szewczuk ◽

Bolesław T. Karwowski

Keyword(s):

Xeroderma Pigmentosum ◽

Excision Repair ◽

Normal Skin ◽

Dna Lesions ◽

Repair Processes ◽

Nuclear Extracts ◽

Cellular Repair ◽

Base Excision ◽

Synthetic Oligonucleotides ◽

Ap Site

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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