DNA-protein crosslink formation by endogenous aldehydes and AP sites

Abstract

Covalent binding between proteins and a DNA strand produces DNA-protein crosslinks (DPC). DPC are one of the most deleterious types of DNA damage, leading to the blockage of DNA replication and transcription. Both DNA lesions and endogenous products with carbonyl functional groups can produce DPC in genomic DNA under normal physiological conditions. For example, formaldehyde, the most abundant endogenous human carcinogen, and apurinic/apyrimidinic (AP) sites, the most common type of endogenous DNA lesions, has been shown to crosslink proteins and/or DNA through their carbonyl functional groups. Unfortunately, compared to other types of DNA damage, DPC have been less studied and understood. However, a recent advancement has allowed researchers to determine accurate yields of various DNA lesions including formaldehyde-derived DPC with high sensitivity and specificity, paving the way for new developments in this field of research. Here, we review the current literature and remaining unanswered questions on DPC formation by endogenous formaldehyde and various aldehydic 2-deoxyribose lesions.

Keywords: AP sites; DPC; Endogenous aldehyde; Formaldehyde; Oxidative 2-deoxyribose.

Fig. 1.. Derivatization Method for Native AP Sites and C1-Oxidized Deoxyriboses.

ARP [25], O-(tetrahydro-2H-pyran-2-yl)hydroxylamine [11], and O-(pyridin-3-yl-methyl)hydroxylamine [23] covalently react with native aldehydic AP sites through the O-hydroxylamine moiety. Pentafluorophenyl hydrazine covalently binds to 5-methylene-2(5H)-furanone derived from 2-deoxyribonolactone [22].

Fig. 2.. Native AP site chemical reactions forming N-CH₂-N and N-CH₂-S linkages from FA, proteins, and nucleobases.

Native AP sites are in equilibrium between the ring-closed form and the ring-opened aldehydic form. The latter can reversibly bind to the amino group of proteins (green) via a Schiff base formation, leading to the formation of an unstable DPC. Subsequently, there is a cleavage reaction of the DNA strand through β-elimination, after which the protein can be dissociated from the 3’-AP sites. Alternatively, DPC can be formed between native aldehydic AP sites and the N-terminal Cys of proteins (blue). The AP sites can reversibly bind to the α-amino group of proteins via a Schiff base formation, leading to DPC formation. The DPC then rearranges to produce a reversible thiazolidine linkage between the AP site, the α-amino group, and the sulfhydryl moiety of its amino-terminal Cys.

Fig. 3.. Structures of a nucleoside and the major aldehydic/ketonic oxidized 2’-deoxyriboses attached to a phosphodiester backbone

(modified from [24] with permission)

Fig. 4.. α,-β Unsaturated aldehyde leading to aldehydic base lesions.

Lipid peroxidation-derived α,-β unsaturated aldehydes produce dG adducts, which are in equilibrium between the ring-opened aldehydic form and the ring-closed exocyclic form (modified from [53] with permission)

Fig. 5.. Tumor and DPC formation in the nasal tissue of animals exposed to formaldehyde.

A. Nasal tumor (SCC) incidence in rats exposed to formaldehyde by inhalation for 18 and 24 months (6 hour/day, 5 days/week) [61,65]. B. Endogenous and exogenous FA-induced DPC (dG-Me-Cys) in the nasal tissue of rats exposed to air control vs. 15 ppm of [13CD2]-formaldehyde (6 hour/day) [9]. C. Endogenous and exogenous FA-induced DPC (dG-Me-Cys) in the nasal tissue of rats exposed to 2 ppm of [13CD2]-formaldehyde (6 hour/day, 5 days/week) [9]. D. Effects of exogenous FA exposure on endogenous FA-derived DPC (dG-Me-Cys) in rats exposed to air control vs. 15 ppm of [13CD2]-formaldehyde for 4 consecutive days (6 hour/day) [9] E. Endogenous and exogenous FA-induced DPC (dG-Me-Cys) in the nose and bone marrow of non-human primates (NHP, Cynomolgus Macaque) exposed to air control vs. 6 ppm of [13CD2]-formaldehyde for 2 days (6 hour/day) [9].

Fig. 6.. Chemical reactions forming N-CH₂-N and N-CH₂-S linkages from FA, protein, and nucleobase.

A. N-CH₂-N linkage: FA reacts with the amino group of proteins, resulting in the formation of methylol adducts on proteins. After dehydration, the Schiff base further binds to the nucleophilic amino moiety of the nucleobase through a methylene linkage (N-CH₂-N). B. N-CH₂-S linkage: FA first binds to the thiol group of Cys in proteins, leading to methylol adducts on the protein. The methylol adducts react with nucleobases after dehydration. All of the reactions (A and B) are reversible (modified from [83,103] with permission).

Fig. 7.. Endogenous FA-derived dG mono adducts vs DPC in tissues/organs of rats and monkeys.

A. Endogenous FA-derived dG mono adducts (N2-HOMe-dG adducts/10⁷ dG) in rat tissues [68]. B. Endogenous FA-derived DPC (dG-Me-Cys/10⁷ dG) in rat tissues [68]. C. Endogenous FA-derived DPC/dG mono adduct ratio in rat tissues [68]. D. Endogenous FA-derived DPC (dG-Me-Cys/10⁷ dG) in non-human primate (NHP) tissues [9].

Fig. 8.. Structures of M2FA-Lys and M2FA-dA.

A. 1,4-dihydropyridine-type malondialdehyde (MDA)-FA-Lys (M2FA-Lys) adducts are formed by a reaction between FA (red) and two equivalents of MDA (blue) with a Lys (black) [72]. B. Likewise, incubation of FA, MDA, and dA produces M2FA-dA [98].