Nathan E. Price

Interstrand DNA–DNA Cross-Link Formation Between Adenine Residues and Abasic Sites in Duplex DNA

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 N6-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.

Chemical and structural characterization of interstrand cross-links formed between abasic sites and adenine residues in duplex DNA

A new type of interstrand DNA–DNA cross-link between abasic (Ap) sites and 2′-deoxyadenosine (dA) residues was recently reported, but the chemical structure and properties of this lesion were not rigorously established. Here we characterized the nucleoside cross-link remnant released by enzymatic digestion of duplex DNA containing the dA-Ap cross-link. A synthetic standard was prepared for the putative nucleoside cross-link remnant 6 in which the anomeric carbon of the 2-deoxyribose residue was connected to the exocyclic N6-amino group of dA. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the synthetic material 6 matched the authentic cross-link remnant released by enzymatic digestion of cross-linked DNA. These findings establish the chemical structure of the dA-Ap cross-link released from duplex DNA and may provide methods for the detection of this lesion in cellular DNA. Both the nucleoside cross-link remnant 6 and the cross-link in duplex DNA were quite stable at pH 7 and 37°C, suggesting that the dA-Ap cross-link could be a persistent lesion with the potential to block the action of various DNA processing enzymes.

Characterization of interstrand DNA-DNA cross-links derived from abasic sites using bacteriophage ϕ29 DNA polymerase

Interstrand cross-links in cellular DNA are highly deleterious lesions that block transcription and replication. We recently characterized two new structural types of interstrand cross-links derived from the reaction of abasic (Ap) sites with either guanine or adenine residues in duplex DNA. Interestingly, these Ap-derived cross-links are forged by chemically reversible processes, in which the two strands of the duplex are joined by hemiaminal, imine, or aminoglycoside linkages. Therefore, understanding the stability of Ap-derived cross-links may be critical in defining the potential biological consequences of these lesions. Here we employed bacteriophage φ29 DNA polymerase, which can couple DNA synthesis and strand displacement, as a model system to examine whether dA-Ap cross-links can withstand DNA-processing enzymes. We first demonstrated that a chemically stable interstrand cross-link generated by hydride reduction of the dG-Ap cross-link completely blocked primer extension by φ29 DNA polymerase at the last unmodified nucleobase preceding cross-link. We then showed that the nominally reversible dA-Ap cross-link behaved, for all practical purposes, like an irreversible, covalent DNA-DNA cross-link. The dA-Ap cross-link completely blocked progress of the φ29 DNA polymerase at the last unmodified base before the cross-link. This suggests that Ap-derived cross-links have the power to block various DNA-processing enzymes in the cell. In addition, our results reveal φ29 DNA polymerase as a tool for detecting the presence and mapping the location of interstrand cross-links (and possibly other lesions) embedded within regions of duplex DNA.

Characterization of Interstrand DNA–DNA Cross-Links Using the α-Hemolysin Protein Nanopore

Nanopore-based sensors have been studied extensively as potential tools for DNA sequencing, characterization of epigenetic modifications such as 5-methylcytosine, and detection of microRNA biomarkers. In the studies described here, the α-hemolysin protein nanopore embedded in a lipid bilayer was used for the detection and characterization of interstrand cross-links in duplex DNA. Interstrand cross-links are important lesions in medicinal chemistry and toxicology because they prevent the strand separation that is required for read-out of genetic information from DNA in cells. In addition, interstrand cross-links are used for the stabilization of duplex DNA in structural biology and materials science. Cross-linked DNA fragments produced unmistakable current signatures in the nanopore experiment. Some cross-linked substrates gave irreversible current blocks of >10 min, while others produced long current blocks (10-100 s) before the double-stranded DNA cross-link translocated through the α-hemolysin channel in a voltage-driven manner. The duration of the current block for the different cross-linked substrates examined here may be dictated by the stability of the duplex region left in the vestibule of the nanopore following partial unzipping of the cross-linked DNA. Construction of calibration curves measuring the frequency of cross-link blocking events (1/τon) as a function of cross-link concentration enabled quantitative determination of the amounts of cross-linked DNA present in samples. The unique current signatures generated by cross-linked DNA in the α-HL nanopore may enable the detection and characterization of DNA cross-links that are important in toxicology, medicine, and materials science.

Thiol-activated DNA damage by α-bromo-2-cyclopentenone.

Some biologically active chemicals are relatively stable in the extracellular environment but, upon entering the cell, undergo biotransformation into reactive intermediates that covalently modify DNA. The diverse chemical reactions involved in the bioactivation of DNA-damaging agents are both fundamentally interesting and of practical importance in medicinal chemistry and toxicology. The work described here examines the bioactivation of α-haloacrolyl-containing molecules. The α-haloacrolyl moiety is found in a variety of cytotoxic natural products including clionastatin B, bromovulone III, discorahabdins A, B, and C, and trichodenone C, in mutagens such as 2-bromoacrolein and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), and in the anticancer drug candidates brostallicin and PNU-151807. Using α-bromo-2-cyclopentenone (1) as a model compound, the activation of α-haloacrolyl-containing molecules by biological thiols was explored. The results indicate that both low molecular weight and peptide thiols readily undergo conjugate addition to 1. The resulting products are consistent with a mechanism in which initial addition of thiols to 1 is followed by intramolecular displacement of bromide to yield a DNA-alkylating episulfonium ion intermediate. The reaction of thiol-activated 1 with DNA produces labile lesions at deoxyguanosine residues. The sequence specificity and salt dependence of this process is consistent with involvement of an episulfonium ion intermediate. The alkylated guanine residue resulting from the thiol-triggered reaction of 1 with duplex DNA was characterized using mass spectrometry. The results provide new insight regarding the mechanisms by which thiols can bioactivate small molecules and offer a more complete understanding of the molecular mechanisms underlying the biological activity of cytotoxic, mutagenic, and medicinal compounds containing the α-haloacrolyl group.

Covalent adduct formation between the antihypertensive drug hydralazine and abasic sites in double- and single-stranded DNA.

Hydralazine (4) is an antihypertensive agent that displays both mutagenic and epigenetic properties. Here, gel electrophoretic, mass spectroscopic, and chemical kinetics methods were used to provide evidence that medicinally relevant concentrations of 4 rapidly form covalent adducts with abasic sites in double- and single-stranded DNA under physiological conditions. These findings raise the intriguing possibility that the genotoxic properties of this clinically used drug arise via reactions with an endogenous DNA lesion rather than with the canonical structure of DNA.

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

Abstract 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.

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

Abstract 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) η 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 κ, Pol ι or Pol ζ, 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.