Structural Characterization of Formaldehyde-Induced Cross-Links Between Amino Acids and Deoxynucleosides and Their Oligomers

Abstract

Exposure to formaldehyde results in the formation of DNA-protein cross-links (DPCs) as a primary genotoxic effect. Although DPCs are biologically important and eight amino acids have been reported to form stable adducts with formaldehyde, the structures of these cross-links have not yet been elucidated. We have characterized formaldehyde-induced cross-links of Lys, Cys, His and Trp with dG, dA and dC. dT formed no cross-links, nor did Arg, Gln, Tyr or Asn. Reaction of formaldehyde with Lys and dG gave the highest yield of cross-linked products, followed by reaction with Cys and dG. Yields from the other coupling reactions were lower by a factor of 10 or more. Detailed structural examination by NMR and mass spectrometry established that the cross-links between amino acids and single nucleosides involve a formaldehyde-derived methylene bridge. Lys yielded two additional products with dG in which the linking structure is a 1,N2-fused triazino ring. The Lys cross-linked products were unstable at ambient temperature. Reactions between the reactive Nα-Boc-protected amino acids and the trinucleotides d(T1B2T3) where B2 is the target base G, A or C and reactions between dG, dA and dC and 8-mer peptides containing a single reactive target residue at position 5 yielded cross-linked products with structures inferred from high resolution mass spectrometry and fragmentation patterns that are consistent with those between Nα-Boc-protected amino acids and single nucleotides rigorously determined by NMR studies. These structures will provide a basis for investigation of the characteristics and properties of DPCs formed in vivo and will be helpful in identifying biomarkers for the evaluation of formaldehyde exposure both at site of contact and at distant sites.

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

“…In comparing the products of reactions containing lysine, cysteine, histidine, or tryptophan with each of the four DNA bases, the highest yield of crosslinked product was obtained with lysine and deoxyguanosine (51), consistent with lysine being the most reactive among residues in native proteins as described above. Similar results were obtained using short peptides and trinucleotides (51). In the context of protein-DNA interactions, the first chemical step could involve reaction with an amino acid side chain in a protein, the protein N terminus, or an amino or imino group on a DNA base; importantly, however, the ⑀-amino group on the lysine side chain is a better nucleophile than are the amino/imino groups on DNA bases whose lone pair electrons are delocalized in the aromatic ring.…”

Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage

“…Characterization and In Vitro Degradation of Formaldehyde-Induced DPCs Based on previous work in our lab (Lu et al, , 2010b, dG-Me-GSH and dG-Me-AGT are formed in vitro and contain specific dG-Me-Cysteine linkages (Fig. 1A).…”

“…Previous in vitro studies demonstrated DPCs were induced by formaldehyde with formation of N-CH 2 -N and N-CH 2 -S linkages between DNA and protein (Heck et al, 1990). To study the chemical identity of formaldehyde-induced DPCs, we investigated the in vitro crosslinking reaction between nucleosides, nucleotides, and amino acids and peptides in the presence of formaldehyde (Lu et al, 2010b). Our results demonstrate that the most abundant crosslink was formed with N-CH 2 -N linkage between dG and lysine.…”

“…However, it has not been possible to know the chemical identity of formaldehyde-induced DPCs formed in vivo due to the lack of chemical specific and accurate measurement. In this study, dG-Me-Cys containing N-CH 2 -S linkage was chosen for the degradation experiments based on its relatively high stability and abundance (Lu et al, 2010b). This study also included degradation of other crosslinks, dGMe-GSH, and dG-Me-AGT, both containing N-CH 2 -S linkage.…”

“…The synthesis of dG-Me-GSH was carried out according to a previous report (Lu et al, 2010b). Briefly, glutathione (85 mM) was incubated with formaldehyde (100 mM) in 1.75 ml sodium phosphate buffer (100 mM, pH 7.2) at 37 C for 4 h. dG was then added to a final concentration of 16 mM.…”