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.

UNG-1 and APN-1 are the major enzymes to efficiently repair 5-hydroxymethyluracil DNA lesions in C. elegans

“…We exclude the possibility that EXO-3 acts to remove the 3′- α, β unsaturated aldehyde generated by NTH-1, as the exo-3(tm4374); apn-1(RNAi) knockdown mutant is no more sensitive to 5-hmU than the apn-1(tm6691) single mutant. Consistent with this notion, we have previously shown that EXO-3, unlike APN-1, lacks a 3′- to 5′-exonuclease activity, which might be the activity needed to remove such bulky and toxic 3′-blocked end 18 – 21 . In fact, it has been reported that diminishing UNG-1 activity to prevent production of AP sites recues the lifespan defect of the exo-3 mutants 23 .…”

“…This 3′-blocking lesion must be removed by one of the two conserved AP endonucleases/3′-diesterases, APN-1 and EXO-3, to produce a 3′-hydroxyl group for DNA repair synthesis 18 – 20 . If the 3′-blocking lesions are not efficiently removed, they can also generate DNA and protein crosslinks that become more deleterious than simple abasic sites 21 .…”

Interaction of Functionalized Naphthalenophanes with Abasic Sites in DNA: DNA Cleavage, DNA Cleavage Inhibition, and Formation of Ligand–DNA Adducts

“…Although the reaction of β‐elimination products of AP‐site cleavage with external alkoxyamines is known and has been exploited for their quantitative detection, the formation of covalent adducts with 2,7‐BisNP‐NH represents, to the best of our knowledge, the first example of the generation of stable adducts through the reaction of cleaved AP sites with polyamine ligands. In this context, it is interesting to mention the formation of stable interstrand cross‐links through the reaction of both uncleaved and cleaved AP sites with purine residues observed under native (nonreducing) conditions. Notably, in the latter case, the formation of the cross‐link was proposed to occur through a 1,4‐conjugate addition of N1 of a proximate adenine residue to the unsaturated aldehyde product of β‐elimination cleavage, which is reminiscent of the mechanism proposed above.…”

“…In analogy to other intrastrand and smallmolecule cross-links of AP sites, we expect this adduct to be genotoxic, representing ah urdle for DNA polymerases. [50,52,56] It may also be potentially harnessed for covalent targeting of AP sites and incorporation of reporter moieties, such as fluorophores or affinity tags for DNA pull-downstudies.…”

Homocysteine aggravates DNA damage by impairing the FA/Brca1 Pathway in NE4C murine neural stem cells

“…Interstrand cross-links (ICLs) are severe DNA lesions induced by numerous agents, including mitomycin C (MMC), cisplatin and endogenous products of lipid peroxidation [23,24]. By covalently linking both DNA strands, ICLs are highly toxic, as they can physically block DNA melting, transcription, and replication [25].…”