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Interstrand Cross-Link Formation Involving Reaction of a Mispaired Cytosine Residue with an Abasic Site in Duplex DNA.

ABSTRACT: The formation of interstrand cross-links in duplex DNA is important in biology, medicine, and biotechnology. Interstrand cross-links arising from the reaction of the aldehyde residue of an abasic (apurinic or AP) site with the exocyclic amino groups of guanine or adenine residues on the opposing strand of duplex DNA have previously been characterized. The canonical nucleobase cytosine has an exocyclic amino group but its ability to form interstrand cross-links by reaction with an AP site has not been characterized before now. Here it is shown that substantial yields of interstrand cross-links are generated in sequences having a mispaired cytosine residue located one nucleotide to the 3'-side of the AP site on the opposing strand (e.g., 5'XA/5'CA, where X = AP). Formation of the dC-AP cross-link is pH-dependent, with significantly higher yields at pH 5 than pH 7. Once formed, the dC-AP cross-link is quite stable, showing less than 5% dissociation over the course of 96 h at pH 7 and 37 °C. No significant yields of cross-link are observed when the cytosine residue is paired with its Watson-Crick partner guanine. It was also shown that a single AP site can engage with multiple nucleobase cross-linking partners in some sequences. Specifically, the dG-AP and dC-AP cross-links coexist in dynamic equilibrium in the sequence 5'CXA/5'CAG (X = AP). In this sequence, the dC-AP cross-link dominates. However, in the presence of NaBH₃CN, irreversible reduction of small amounts of the dG-AP cross-link present in the mixture shifts the equilibria away from the dC-AP cross-link toward good yields of the dG-AP_red cross-link.

SUBMITTER: Varela JG

PROVIDER: S-EPMC8650171 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Interstrand DNA Cross-Links Derived from Reaction of a 2-Aminopurine Residue with an Abasic Site.

Project description:Efficient methods for the site-specific installation of structurally defined interstrand cross-links in duplex DNA may be useful in a wide variety of fields. The work described here developed a high-yield synthesis of chemically stable interstrand cross-links resulting from a reductive amination reaction between an abasic site and the noncanonical nucleobase 2-aminopurine in duplex DNA. Results from footprinting, liquid chromatography-mass spectrometry, and stability studies support the formation of an N2-alkylamine attachment between the 2-aminopurine residue and the Ap site. The reaction performs best when the 2-aminopurine residue on the opposing strand is offset 1 nt to the 5'-side of the abasic site. The cross-link confers substantial resistance to thermal denaturation (melting). The cross-linking reaction is fast (complete in 4 h), employs only commercially available reagents, and can be used to generate cross-linked duplexes in sufficient quantities for biophysical, structural, and DNA repair studies.

| S-EPMC8667195 | biostudies-literature

Interstrand DNA-DNA cross-link formation between adenine residues and abasic sites in duplex DNA.

Project description:The loss of a coding nucleobase from the structure of DNA is a common event that generates an abasic (Ap) site (1). Ap sites exist as an equilibrating mixture of a cyclic hemiacetal and a ring-opened aldehyde. Aldehydes are electrophilic functional groups that can form covalent adducts with nucleophilic sites in DNA. Thus, Ap sites present a potentially reactive aldehyde as part of the internal structure of DNA. Here we report evidence that the aldehyde group of Ap sites in duplex DNA can form a covalent adduct with the N(6)-amino group of adenine residues on the opposing strand. The resulting interstrand DNA-DNA cross-link occurs at 5'-ApT/5'-AA sequences in remarkably high yields (15-70%) under physiologically relevant conditions. This naturally occurring DNA-templated reaction has the potential to generate cross-links in the genetic material of living cells.

| S-EPMC3954461 | biostudies-literature

Effects of Local Sequence, Reaction Conditions, and Various Additives on the Formation and Stability of Interstrand Cross-Links Derived from the Reaction of an Abasic Site with an Adenine Residue in Duplex DNA.

Project description:The experiments described here examined the effects of reaction conditions, various additives, and local sequence on the formation and stability interstrand cross-links (ICLs) derived from the reaction of an apurinic/apyrimidinic (AP) site with the exocyclic amino group of an adenine residue on the opposing strand in duplex DNA. Cross-link formation was observed in a range of different buffers, with faster formation rates observed at pH 5. Inclusion of the base excision repair enzyme alkyladenine DNA glycosylase (hAAG) which binds tightly to AP-containing duplexes decreased, but did not completely prevent, formation of the dA-AP ICL. Formation of the dA-AP ICL was not altered by the presence of the biological metal ion Mg2+ or the biological thiol, glutathione. Several organocatalysts of imine formation did not enhance the rate of dA-AP ICL formation. Duplex length did not have a large effect on dA-AP yield, so long as the melting temperature of the duplex was not significantly below the reaction temperature (the duplex must remain hybridized for efficient ICL formation). Formation of the dA-AP ICL was examined in over 40 different sequences that varied the neighboring and opposing bases at the cross-linking site. The results indicate that ICL formation can occur in a wide variety of sequence contexts under physiological conditions. Formation of the dA-AP ICL was strongly inhibited by the aldehyde-trapping agents methoxyamine and hydralazine, by NaBH3CN, by the intercalator ethidium bromide, and by the minor groove-binding agent netropsin. ICL formation was inhibited to some extent in bicarbonate and Tris buffers. The dA-AP ICL showed substantial inherent stability under a variety of conditions and was not a substrate for AP-processing enzymes APE1 or Endo IV. Finally, we characterized cross-link formation in a small (11 bp) stem-loop (hairpin) structure and in DNA-RNA hybrid duplexes.

| S-EPMC9583646 | biostudies-literature

On the formation and properties of interstrand DNA-DNA cross-links forged by reaction of an abasic site with the opposing guanine residue of 5'-CAp sequences in duplex DNA.

Project description:We recently reported that the aldehyde residue of an abasic (Ap) site in duplex DNA can generate an interstrand cross-link via reaction with a guanine residue on the opposing strand. This finding is intriguing because the highly deleterious nature of interstrand cross-links suggests that even small amounts of Ap-derived cross-links could make a significant contribution to the biological consequences stemming from the generation of Ap sites in cellular DNA. Incubation of 21-bp duplexes containing a central 5'-CAp sequence under conditions of reductive amination (NaCNBH(3), pH 5.2) generated much higher yields of cross-linked DNA than reported previously. At pH 7, in the absence of reducing agents, these Ap-containing duplexes also produced cross-linked duplexes that were readily detected on denaturing polyacrylamide gels. Cross-link formation was not highly sensitive to reaction conditions, and the cross-link, once formed, was stable to a variety of workup conditions. Results of multiple experiments including MALDI-TOF mass spectrometry, gel mobility, methoxyamine capping of the Ap aldehyde, inosine-for-guanine replacement, hydroxyl radical footprinting, and LC-MS/MS were consistent with a cross-linking mechanism involving reversible reaction of the Ap aldehyde residue with the N(2)-amino group of the opposing guanine residue in 5'-CAp sequences to generate hemiaminal, imine, or cyclic hemiaminal cross-links (7-10) that were irreversibly converted under conditions of reductive amination (NaCNBH(3)/pH 5.2) to a stable amine linkage. Further support for the importance of the exocyclic N(2)-amino group in this reaction was provided by an experiment showing that installation of a 2-aminopurine-thymine base pair at the cross-linking site produced high yields (15-30%) of a cross-linked duplex at neutral pH, in the absence of NaCNBH(3).

| S-EPMC4012394 | biostudies-literature

Scope and mechanism of interstrand cross-link formation by the C4'-oxidized abasic site.

Project description:The C4'-oxidized abasic site (C4-AP) is a commonly formed DNA lesion, which generates two types of interstrand cross-links (ICLs). The kinetically favored cross-link consists of two full length strands and forms reversibly and exclusively with dA. Cross-link formation is attributed to condensation of C4-AP with the N6-amino group of dA. Formation of the thermodynamic ICL involves cleavage of the strand containing C4-AP on the 3'-side of the lesion. The ratios and yields of the ICLs are highly dependent upon the local sequence. Product analysis of enzyme-digested material reveals that the ICL with dA is a cyclic adduct. Formation of the thermodynamically favored cross-link is catalyzed by the surrounding DNA sequence and occurs favorably with dC and dA but not with dG or dT. Mechanistic studies indicate that beta-elimination from C4-AP is the rate-limiting step in the formation of the thermodynamic ICL and that the local DNA environment determines the rate constant for this reaction. The efficiency of ICL formation, the stability of the thermodynamic products, and their possible formation in cells (Regelus, P.; et al. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14032) suggest that these lesions will be deleterious to the biological system in which they are produced.

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Replication and repair of a reduced 2?-deoxyguanosine-abasic site interstrand cross-link in human cells.

Project description:Apurinic/apyrimidinic (AP) sites, or abasic sites, which are a common type of endogenous DNA damage, can forge interstrand DNA-DNA cross-links via reaction with the exocyclic amino group on a nearby 2?-deoxyguanosine or 2?-deoxyadenosine in the opposite strand. Here, we utilized a shuttle vector method to examine the efficiency and fidelity with which a reduced dG-AP cross-link-containing plasmid was replicated in cultured human cells. Our results showed that the cross-link constituted strong impediments to DNA replication in HEK293T cells, with the bypass efficiencies for the dG- and AP-containing strands being 40% and 20%, respectively. While depletion of polymerase (Pol) ? did not perturb the bypass efficiency of the lesion, the bypass efficiency was markedly reduced (to 1-10%) in the isogenic cells deficient in Pol ?, Pol ? or Pol ?, suggesting the mutual involvement of multiple translesion synthesis polymerases in bypassing the lesion. Additionally, replication of the cross-linked AP residue in HEK293T cells was moderately error-prone, inducing a total of ?26% single-nucleobase substitutions at the lesion site, whereas replication past the cross-linked dG component occurred at a mutation frequency of ?8%. Together, our results provided important insights into the effects of an AP-derived interstrand cross-link on the efficiency and accuracy of DNA replication in human cells.

| S-EPMC5499640 | biostudies-literature

Chemical structure and properties of interstrand cross-links formed by reaction of guanine residues with abasic sites in duplex DNA.

Project description:A new type of interstrand cross-link resulting from the reaction of a DNA abasic site with a guanine residue on the opposing strand of the double helix was recently identified, but the chemical connectivity of the cross-link was not rigorously established. The work described here was designed to characterize the chemical structure and properties of dG-AP cross-links generated in duplex DNA. The approach involved characterization of the nucleoside cross-link "remnant" released by enzymatic digestion of DNA duplexes containing the dG-AP cross-link. We first carried out a chemical synthesis and complete spectroscopic structure determination of the putative cross-link remnant 9b composed of a 2-deoxyribose adduct attached to the exocyclic N(2)-amino group of dG. A reduced analogue of the cross-link remnant was also prepared (11b). Liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis revealed that the retention times and mass spectral properties of synthetic standards 9b and 11b matched those of the authentic cross-link remnants released by enzymatic digestion of duplexes containing the native and reduced dG-AP cross-link, respectively. These results establish the chemical connectivity of the dG-AP cross-link released from duplex DNA and provide a foundation for detection of this lesion in biological samples. The dG-AP cross-link in duplex DNA was remarkably stable, decomposing with a half-life of 22 days at pH 7 and 23 °C. The intrinsic chemical stability of the dG-AP cross-link suggests that this lesion in duplex DNA may have the power to block DNA-processing enzymes involved in transcription and replication.

| S-EPMC4706233 | biostudies-literature

DNA interstrand cross-link formation by the 1,4-dioxobutane abasic lesion.

Project description:The oxidized abasic lesion 5'-(2-phosphoryl-1,4-dioxobutane) (DOB) is produced concomitantly with a single-strand break by a variety of DNA-damaging agents that abstract a hydrogen atom from the C5'-position. Independent generation of the DOB lesion in DNA reveals that it reversibly forms interstrand cross-links (ICLs) selectively with a dA opposite the 3'-adjacent nucleotide. Product studies and the use of monoaldehyde models suggest that ICL formation involves condensation of the dialdehyde with the exocyclic amine. Mechanistic studies and inspection of molecular models indicate that the local DNA environment and proximity of the exocyclic amine determine the selectivity for reaction with dA. Proximity control of the electrophile's reactivity is distinct from that of structurally similar freely diffusing molecules. ICL formation by a DOB lesion that is adjacent to a single-strand break is potentially significant because the product constitutes a "clustered" or "complex" lesion. Clustered lesions can lead to highly deleterious double-strand breaks upon nucleotide excision repair.

| S-EPMC2784881 | biostudies-literature

Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA.

Project description:Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3'ddR5p) at the 3'-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3'ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3'ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems.

| S-EPMC5499897 | biostudies-literature

Base excision repair enzymes protect abasic sites in duplex DNA from interstrand cross-links.

Project description:Hydrolysis of the N-glycosyl bond between a nucleobase and deoxyribose leaves an abasic site within duplex DNA. The abasic site can react with exocyclic amines of nucleobases on the complementary strand to form interstrand DNA-DNA cross-links (ICLs). We find that several enzymes from the base excision repair (BER) pathway protect an abasic site on one strand of a DNA duplex from cross-linking with an amine on the opposing strand. Human alkyladenine DNA glycosylase (AAG) and Escherichia coli 3-methyladenine DNA glycosylase II (AlkA) accomplish this by binding tightly to the abasic site and sequestering it. AAG protects an abasic site opposite T, the product of its canonical glycosylase reaction, by a factor of ?10-fold, as estimated from its inhibition of the reaction of an exogenous amine with the damaged DNA. Human apurinic/apyrimidinic site endonuclease 1 and E. coli endonuclease III both decrease the amount of ICL at equilibrium by generating a single-strand DNA nick at the abasic position as it is liberated from the cross-link. The reversibility of the reaction between amines and abasic sites allows BER enzymes to counter the potentially disruptive effects of this type of cross-link on DNA transactions.

| S-EPMC4404639 | biostudies-other

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