Repurposing the Antihypertensive Agent Hydralazine As an Inhibitor of the Base Excision Repair Enzyme APE1.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a central enzyme in the base excision repair (BER) pathway. APE1 catalyzes incision of the phosphodiester linkage on the 5'-side of apurinic/apyrimidinic (AP) sites during the repair of damaged nucleobases in cellular DNA. Inhibition of this enzyme can potentiate the action of DNA-damaging chemotherapeutic agents.

The Intercalator Ethidium Bromide Generates Covalent Adducts at Apurinic/Apyrimidinic Sites in DNA.

Ethidium bromide was first described as a DNA intercalator 60 years ago and, over the ensuing years, may be the most widely used fluorescent DNA stain in molecular biology, biochemistry, and histology. Noncovalent DNA binding by ethidium has been well characterized, but to date, there have been no reports of covalent DNA adduct formation by ethidium bromide. This report describes the characterization of covalent adducts generated by the reaction of ethidium with apurinic/apyrimidinic (AP) sites in DNA.

Noncovalent Inhibition and Covalent Inactivation of Proline Dehydrogenase by Analogs of -Propargylglycine.

The flavoenzyme proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the oxidation of l-proline to Δ-pyrroline-5-carboxylate. The enzyme is a target for chemical probe discovery because of its role in the metabolism of certain cancer cells. -propargylglycine is the first and best characterized mechanism-based covalent inactivator of PRODH.

Novel Processes Associated with the Repair of Interstrand Cross-Links Derived from Abasic Sites in Duplex DNA: Roles for the Base Excision Repair Glycosylase NEIL3 and the SRAP Protein HMCES.

Recent studies have defined a novel pathway for the repair of interstrand cross-links derived from the reaction of an adenine residue with an apurinic/apyrimidinic (AP) site on the opposing strand of DNA (dA-AP ICL). Stalling of a replication fork at the dA-AP ICL triggers TRAIP-dependent ubiquitylation of the CMG helicase that recruits the base excision repair glycosylase NEIL3 to the lesion. NEIL3 unhooks the dA-AP ICL to regenerate the native adenine residue on one strand and an AP site on the other strand.

Formation and Properties of DNA Adducts Generated by Reactions of Abasic Sites with 1,2-Aminothiols Including Cysteamine, Cysteine Methyl Ester, and Peptides Containing -Terminal Cysteine Residues.

The reaction of 1,2-aminothiol groups with aldehyde residues in aqueous solution generates thiazolidine products, and this process has been developed as a catalyst-free click reaction for bioconjugation. The work reported here characterized reactions of the biologically relevant 1,2-aminothiols including cysteamine, cysteine methyl ester, and peptides containing -terminal cysteine residues with the aldehyde residue of apurinic/apyrimidinic (AP) sites in DNA oligomers. These 1,2-aminothiol-containing compounds rapidly generated adducts with AP sites in single-stranded and double-stranded DNA.

-Methyl--nitrosourea Induced 3'-Glutathionylated DNA-Cleavage Products in Mammalian Cells.

Apurinic/apyrimidinic (AP) sites, that is, abasic sites, are among the most frequently induced DNA lesions. Spontaneous or DNA glycosylase-mediated β-elimination of the 3'-phosphoryl group can lead to strand cleavages at AP sites to yield a highly reactive, electrophilic 3'-phospho-α,β-unsaturated aldehyde (3'-PUA) remnant. The latter can react with amine or thiol groups of biological small molecules, DNA, and proteins to yield various damaged 3'-end products.

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.

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.

Reconsidering the Chemical Nature of Strand Breaks Derived from Abasic Sites in Cellular DNA: Evidence for 3'-Glutathionylation.

The hydrolytic loss of coding bases from cellular DNA is a common and unavoidable reaction. The resulting abasic sites can undergo β-elimination of the 3'-phosphoryl group to generate a strand break with an electrophilic α,β-unsaturated aldehyde residue on the 3'-terminus. The work reported here provides evidence that the thiol residue of the cellular tripeptide glutathione rapidly adds to the alkenal group on the 3'-terminus of an AP-derived strand break.

Unexpected Complexity in the Products Arising from NaOH-, Heat-, Amine-, and Glycosylase-Induced Strand Cleavage at an Abasic Site in DNA.

Hydrolytic loss of nucleobases from the deoxyribose backbone of DNA is one of the most common unavoidable types of damage in synthetic and cellular DNA. The reaction generates abasic sites in DNA, and it is important to understand the properties of these lesions. The acidic nature of the α-protons of the ring-opened abasic aldehyde residue facilitates the β-elimination of the 3'-phosphoryl group.

Products Generated by Amine-Catalyzed Strand Cleavage at Apurinic/Apyrimidinic Sites in DNA: New Insights from a Biomimetic Nucleoside Model System.

Abasic sites are common in cellular and synthetic DNA. As a result, it is important to characterize the chemical fate of these lesions. Amine-catalyzed strand cleavage at abasic sites in DNA is an important process in which conversion of small amounts of the ring-opened abasic aldehyde residue to an iminium ion facilitates β-elimination of the 3'-phosphoryl group.

Synthesis of DNA Duplexes Containing Site-Specific Interstrand Cross-Links via Sequential Reductive Amination Reactions Involving Diamine Linkers and Abasic Sites on Complementary Oligodeoxynucleotides.

Interstrand DNA cross-links are important in biology, medicinal chemistry, and materials science. Accordingly, methods for the targeted installation of interstrand cross-links in DNA duplexes may be useful in diverse fields. Here, a simple procedure is reported for the preparation of DNA duplexes containing site-specific, chemically defined interstrand cross-links.

Photoinduced Covalent Irreversible Inactivation of Proline Dehydrogenase by S-Heterocycles.

Proline dehydrogenase (PRODH) is a flavoenzyme that catalyzes the first step of proline catabolism, the oxidation of l-proline to Δ-pyrroline-5-carboxylate. PRODH has emerged as a cancer therapy target because of its involvement in the metabolic reprogramming of cancer cells. Here, we report the discovery of a new class of PRODH inactivator, which covalently and irreversibly modifies the FAD in a light-dependent manner.

Formation and Repair of an Interstrand DNA Cross-Link Arising from a Common Endogenous Lesion.

Interstrand DNA cross-links (ICLs) are cytotoxic because they block the strand separation required for read-out and replication of the genetic information in duplex DNA. The unavoidable formation of ICLs in cellular DNA may contribute to aging, neurodegeneration, and cancer. Here, we describe the formation and properties of a structurally complex ICL derived from an apurinic/apyrimidinic (AP) site, which is one of the most common endogenous lesions in cellular DNA.

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

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.

Formation and repair of unavoidable, endogenous interstrand cross-links in cellular DNA.

Genome integrity is essential for life and, as a result, DNA repair systems evolved to remove unavoidable DNA lesions from cellular DNA. Many forms of life possess the capacity to remove interstrand DNA cross-links (ICLs) from their genome but the identity of the naturally-occurring, endogenous substrates that drove the evolution and retention of these DNA repair systems across a wide range of life forms remains uncertain. In this review, we describe more than a dozen chemical processes by which endogenous ICLs plausibly can be introduced into cellular DNA.

Structure of a Stable Interstrand DNA Cross-Link Involving a β--Glycosyl Linkage Between an -dA Amino Group and an Abasic Site.

Abasic (AP) sites are one of the most common forms of DNA damage. The deoxyribose ring of AP sites undergoes anomerization between α and β configurations, via an electrophilic aldehyde intermediate. In sequences where an adenine residue is located on the opposing strand and offset 1 nt to the 3' side of the AP site, the nucleophilic -dA amino group can react with the AP aldehyde residue to form an interstrand cross-link (ICL).

An autoinhibitory role for the GRF zinc finger domain of DNA glycosylase NEIL3.

The NEIL3 DNA glycosylase maintains genome integrity during replication by excising oxidized bases from single-stranded DNA (ssDNA) and unhooking interstrand cross-links (ICLs) at fork structures. In addition to its N-terminal catalytic glycosylase domain, NEIL3 contains two tandem C-terminal GRF-type zinc fingers that are absent in the other NEIL paralogs. ssDNA binding by the GRF-ZF motifs helps recruit NEIL3 to replication forks converged at an ICL, but the nature of DNA binding and the effect of the GRF-ZF domain on catalysis of base excision and ICL unhooking is unknown.

Inhibition, crystal structures, and in-solution oligomeric structure of aldehyde dehydrogenase 9A1.

Aldehyde dehydrogenase 9A1 (ALDH9A1) is a human enzyme that catalyzes the NAD-dependent oxidation of the carnitine precursor 4-trimethylaminobutyraldehyde to 4-N-trimethylaminobutyrate. Here we show that the broad-spectrum ALDH inhibitor diethylaminobenzaldehyde (DEAB) reversibly inhibits ALDH9A1 in a time-dependent manner. Possible mechanisms of inhibition include covalent reversible inactivation involving the thiohemiacetal intermediate and slow, tight-binding inhibition.

Covalent Modification of the Flavin in Proline Dehydrogenase by Thiazolidine-2-Carboxylate.

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent 2-electron oxidation of l-proline to Δ-pyrroline-5-carboxylate. PRODH has emerged as a possible cancer therapy target, and thus the inhibition of PRODH is of interest. Here we show that the proline analogue thiazolidine-2-carboxylate (T2C) is a mechanism-based inactivator of PRODH.

Unhooking of an interstrand cross-link at DNA fork structures by the DNA glycosylase NEIL3.

Interstrand DNA-DNA cross-links (ICLs) are generated by endogenous processes, drugs, and environmental toxins. Understanding the cellular pathways by which various ICLs are repaired is critical to understanding their biological effects. Recent studies showed that replication-dependent repair of an ICL derived from the reaction of an abasic (AP) site with an adenine residue (dA) on the opposing strand of duplex DNA proceeds via a novel mechanism in which the DNA glycosylase NEIL3 unhooks the ICL.

Structural analysis of pathogenic mutations targeting Glu427 of ALDH7A1, the hot spot residue of pyridoxine-dependent epilepsy.

Certain loss-of-function mutations in the gene encoding the lysine catabolic enzyme aldehyde dehydrogenase 7A1 (ALDH7A1) cause pyridoxine-dependent epilepsy (PDE). Missense mutations of Glu427, especially Glu427Gln, account for ~30% of the mutated alleles in PDE patients, and thus Glu427 has been referred to as a mutation hot spot of PDE. Glu427 is invariant in the ALDH superfamily and forms ionic hydrogen bonds with the nicotinamide ribose of the NAD cofactor.

Selective covalent capture of a DNA sequence corresponding to a cancer-driving C>G mutation in the gene by a chemically reactive probe: optimizing a cross-linking reaction with non-canonical duplex structures.

Covalent reactions are used in the detection of various biological analytes ranging from low molecular weight metabolites to protein-protein complexes. The detection of specific nucleic acid sequences is important in molecular biology and medicine but covalent approaches are less common in this field, in part, due to a deficit of simple and reliable reactions for the covalent capture of target sequences. Covalent anchoring can prevent the denaturation (melting) of probe-target complexes and causes signal degradation in typical hybridization-based assays.

Structural and biochemical consequences of pyridoxine-dependent epilepsy mutations that target the aldehyde binding site of aldehyde dehydrogenase ALDH7A1.

In humans, certain mutations in the gene encoding aldehyde dehydrogenase 7A1 are associated with pyridoxine-dependent epilepsy (PDE). Understanding the impact of PDE-causing mutations on the structure and activity of ALDH7A1 could allow for the prediction of symptom-severity and aid the development of patient-specific medical treatments. Herein, we investigate the biochemical and structural consequences of PDE missense mutations targeting residues in the aldehyde substrate binding site: N167S, P169S, A171V, G174V, and W175G.

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

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 -alkylamine attachment between the 2-aminopurine residue and the Ap site.