The Roles of Beta- and Delta-Eliminations in the Repair of AP Sites in DNA

Thermoplastic Nanofluidic Devices for Identifying Abasic Sites in Single DNA Molecules

Lab on a Chip ◽

10.1039/d0lc01038c ◽

2021 ◽

Author(s):

Steven A Soper ◽

Swarnagowri Vaidyanathan ◽

Franklin Uba ◽

Bo Hu ◽

David Kaufman ◽

...

Keyword(s):

Dna Damage ◽

Therapeutic Efficacy ◽

Double Strand Breaks ◽

Dna Molecules ◽

Strand Breaks ◽

Single Dna Molecules ◽

Abasic Sites ◽

Nanofluidic Devices ◽

Ap Sites

DNA damage can take many forms such as double-strand breaks and/or the formation of abasic (apurinic/apyrimidinic; AP) sites. The presence of AP sites can be used to determine therapeutic efficacy...

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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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Repair of abasic sites by mammalian cell extracts

Biochemical Journal ◽

10.1042/bj3040699 ◽

1994 ◽

Vol 304 (3) ◽

pp. 699-705 ◽

Cited By ~ 31

Author(s):

G Frosina ◽

P Fortini ◽

O Rossi ◽

F Carrozzino ◽

A Abbondandolo ◽

...

Keyword(s):

Mammalian Cells ◽

Excision Repair ◽

Chinese Hamster ◽

Pyrimidine Dimer ◽

Repair Synthesis ◽

Repair Patch ◽

Cell Extracts ◽

Pyrimidine Dimers ◽

Ap Sites ◽

Ap Site

Hamster cell extracts that perform repair synthesis on covalently closed circular DNA containing pyrimidine dimers, were used to study the repair of apurinic/apyrimidinic (AP) sites and methoxyamine (MX)-modified AP sites. Plasmid molecules were heat-treated at pH 5 and incubated with MX when required. The amount of damage introduced ranged from 0.2 to 0.9 AP sites/kb. Extracts were prepared from the Chinese hamster ovary CHO-9 cell line and from its derivative, 43-3B clone which is mutated in the nucleotide excision repair (NER) ERCC1 gene. AP and MX-AP sites stimulated repair synthesis by CHO-9 cell extracts. The level of synthesis correlated with the number of lesions and was of similar magnitude to the repair stimulated by 4.3 u.v. photoproducts/kb. Repair of AP and MX-AP sites was faster than the repair of u.v. damage and was independent of ERCC1 gene product. The high level of repair replication was due to a very efficient and rapid incision of plasmids carrying AP or MX-AP sites, performed by abundant AP endonucleases present in the extract. The calculated average repair patch sizes were: 7 nucleotides per AP site; 10 nucleotides per MX-AP site; 28 nucleotides per (6-4) u.v. photoproduct or cyclobutane pyrimidine dimer. The data indicate that AP and MX-AP sites are very efficiently repaired by base-excision repair in mammalian cells and suggest that MX-AP sites may also be processed via alternative repair mechanisms.

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

10.32469/10355/63781 ◽

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.

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Diverse applications of covalent cross-links in duplex DNA: From anti-cancer drugs to the detection of disease-relevant single nucleotide polymorphisms to the synthesis of well-defined substrates for the study of novel DNA repair processes

10.32469/10355/69816 ◽

2018 ◽

Author(s):

◽

Maryam Imani Nejad

Keyword(s):

Excision Repair ◽

Alkylating Agents ◽

Nucleotide Polymorphisms ◽

Cross Link ◽

Single Nucleotide ◽

Synthetic Dna ◽

Interstrand Cross Links ◽

Cross Links ◽

Ap Sites ◽

Cellular Dna

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Abasic (Ap) sites are a common form of DNA lesion that occur endogenously 50,000-200,000 per cell per day in mammals. The alkylation of the guanine and adenine residues by the alkylating agents such as nitrogen mustards also induces the formation of Ap sites in genomic DNA. Our group recently showed that Ap sites can forge DNA-DNA interstrand cross-links in some sequences via reaction of the Ap aldehyde residue with the exocyclic amino groups of nucleobases, such as adenine and guanine, on the opposing strand of the DNA duplex. The earlier work in the group revealed that formation of these covalent bridges between two DNA strands is highly sequence- dependent. Although interstrand cross-links are one of the most deleterious types of cellular DNA damage, the availability of synthetic DNA duplexes containing chemically well-defined, site-specific interstrand cross-links has been proven to be a valuable tool in biological chemistry and medicine. We prepared and characterized a new Ap-derived interstrand cross-link. In another project, we use these remarkable cross-linking reactions for the covalent capture of disease-relevant single nucleotide polymorphism by using a protein nanopore technology. The complex mechanisms underlying cross-link repair in cells and limited availability of stable and defined cross-link are two major reasons why repair pathways of these lesions are not yet well understood. By preparing a variety of Ap-derived cross-links, we studied the role of a base excision repair DNA glycosylase, NEIL3 in unhooking the lesions.

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Molecular basis of abasic site sensing in single-stranded DNA by the SRAP domain of E. coli yedK

Nucleic Acids Research ◽

10.1093/nar/gkz744 ◽

2019 ◽

Vol 47 (19) ◽

pp. 10388-10399 ◽

Cited By ~ 4

Author(s):

Na Wang ◽

Hongyu Bao ◽

Liu Chen ◽

Yanhong Liu ◽

Yue Li ◽

...

Keyword(s):

Molecular Basis ◽

Substrate Binding ◽

Specific Substrate ◽

Abasic Site ◽

E Coli ◽

Abasic Sites ◽

Ap Sites ◽

Ap Site

Abstract HMCES and yedK were recently identified as sensors of abasic sites in ssDNA. In this study, we present multiple crystal structures captured in the apo-, nonspecific-substrate-binding, specific-substrate-binding, and product-binding states of yedK. In combination with biochemical data, we unveil the molecular basis of AP site sensing in ssDNA by yedK. Our results indicate that yedK has a strong preference for AP site-containing ssDNA over native ssDNA and that the conserved Glu105 residue is important for identifying AP sites in ssDNA. Moreover, our results reveal that a thiazolidine linkage is formed between yedK and AP sites in ssDNA, with the residues that stabilize the thiazolidine linkage important for the formation of DNA-protein crosslinks between yedK and the AP sites. We propose that our findings offer a unique platform to develop yedK and other SRAP domain-containing proteins as tools for detecting abasic sites in vitro and in vivo.

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Non-canonical interaction of DNA repair proteins with intact and cleaved AP sites

DNA Repair ◽

10.1016/j.dnarep.2020.102847 ◽

2020 ◽

Vol 90 ◽

pp. 102847

Author(s):

Svetlana Khodyreva ◽

Olga Lavrik

Keyword(s):

Dna Repair ◽

Dna Repair Proteins ◽

Ap Sites

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Uncoupling of the apyrimidinic/apurinic endonucleolytic and 3′→5′ exonucleolytic activities of the Nfo protein of Mycoplasma pneumoniae through mutation of specific amino acid residues

Microbiology ◽

10.1099/mic.0.077578-0 ◽

2014 ◽

Vol 160 (6) ◽

pp. 1087-1100 ◽

Cited By ~ 3

Author(s):

Silvia Estevão ◽

Pieternella E. van der Spek ◽

Annemarie M. C. van Rossum ◽

Cornelis Vink

Keyword(s):

Mycoplasma Pneumoniae ◽

Divalent Cations ◽

Single Point ◽

Dna Recombination ◽

Point Mutations ◽

Mycoplasma Genitalium ◽

Amino Acid Residues ◽

Specific Amino Acid ◽

Cleavage Activity ◽

Ap Sites

The DNA recombination and repair machineries of Mycoplasma pneumoniae and Mycoplasma genitalium were predicted to consist of a set of ~11 proteins. The function of one of these proteins was inferred from its homology with proteins belonging to the Endo IV enzyme family. The members of this family function in the repair of apyrimidinic/apurinic (AP) sites in DNA. As such activity may be crucial in the mycoplasmal life cycle, we set out to study the Endo IV-like proteins encoded by M. pneumoniae and M. genitalium. Both proteins, termed Nfo Mpn and Nfo Mge , respectively, were assessed for their ability to interact with damaged and undamaged DNA. In the absence of divalent cations, both proteins exhibited specific cleavage of AP sites. Surprisingly, the proteins also recognized and cleaved cholesteryl-bound deoxyribose moieties in DNA, showing that these Nfo proteins may also function in repair of large DNA adducts. In the presence of Mg2+, Nfo Mpn and Nfo Mge also showed 3′→5′ exonucleolytic activity. By introduction of 13 single point mutations at highly conserved positions within Nfo Mpn , two major types of mutants could be distinguished: (i) mutants that showed no, or limited, AP cleavage activity in the presence of EDTA, but displayed significant levels of AP cleavage activity in the presence of Mg2+; these mutants displayed no, or very low, exonucleolytic activity; and (ii) mutants that only demonstrated marginal levels of AP site cleavage activity in the presence of Mg2+ and did not show exonucleolytic activity. Together, these results indicated that the AP endonucleolytic activity of the Nfo Mpn protein can be uncoupled from its 3′→5′ exonucleolytic activity.

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