Site‐Specific 5‐Formyl Cytosine Mediated DNA‐Histone Cross‐Links: Synthesis and Polymerase Bypass by Human DNA Polymerase η

Abstract

DNA-protein cross-links (DPCs) between DNA epigenetic mark 5-formylC and lysine residues of histone proteins spontaneously form in human cells. Such conjugates are likely to influence chromatin structure and mediate DNA replication, transcription, and repair, but are challenging to study due to their reversible nature. Here we report the construction of site specific, hydrolytically stable DPCs between 5fdC in DNA and K4 of histone H3 and an investigation of their effects on DNA replication. Our approach employs oxime ligation, allowing for site-specific conjugation of histones to DNA under physiological conditions. Primer extension experiments revealed that histone H3-DNA crosslinks blocked DNA synthesis by hPol h polymerase, but were bypassed following proteolytic processing.Irreversible entrapment of cellular proteins on genomic DNA following exposure to bis-electrophiles, transition metals, and free radical species gives rise to DNA-protein cross-links (DPCs). [1] DPCs are unusually bulky DNA lesions that have the ability to block DNA transactions including transcription, replication, and repair, potentially leading to genomic instability and cell death. [2] DPCs accumulate in the heart and brain tissues with age and are hypothesized to play an important role in aging, cancer and neurodegenerative diseases. [3] Individuals deficient in SPRTN, a critical gene required for DPC repair, develop the Ruijs-Aalfs syndrome, a human autosomal recessive disorder characterized by accelerated aging, genomic instability, and early-onset of hepatocellular carcinoma. [4] Scheme 1. Reversible histone-DNA cross-linking and their stabilization via reduction of the resulting Schiff bases.

Participation of Histones in DNA Damage and Repair within Nucleosome Core Particles: Mechanism and Applications

“…The involvement of lysine residues in DPC formation with 5fC was confirmed by Tretyakova via MS/MS analysis . Recently, the generation of reversible DPCs by 5fC in NCPs has been postulated to play a vital role in regulating genetic expression in eukaryotic cells. − …”

“…5fC is a nucleobase-oxidized lesion, which is generated by UV and γ irradiation of 5-methylcytosine (5mC) in DNA. Recently, it was identified as an intermediate in active DNA demethylation and is believed to play a role in epigenetics. − The aldehyde in 5fC is a potential source of reactivity with histones in NCPs. Indeed, we generated 5fC at position [89] (5fC[89]) in a NCP and found that it yields DPCs .…”

Synthesis and Excision Repair of Site-Specific 3′-End DNA-Histone Cross-Links Derived from Abasic Sites

“…First, the ε-CH 2 of the lysine is replaced by an oxygen atom, which has a minor effect on the size. 17 Second, the latter has an additional triazole moiety. We did a control experiment and demonstrated that this has little effect on the repair.…”

“…Studying the repair of Schiff base DNA-histone DPCs has been impeded due to their reversibility. Tretyakova and colleagues site-specifically conjugated histone H3 to 5fC via oxime ligation to synthesize a stable adduct . Recently, we prepared stabilized 3′-PUA-histone DPCs (Figure E) by reductive amination and found that they can be excised by three human nucleases, AP endonuclease 1 (APE1), tyrosyl-DNA phosphodiesterase 1 (TDP1), and three-prime repair exonuclease 1 (TREX1) with moderate efficiency .…”

“…Compared to Schiff base 3′-PUA-histone DPC, the linkage in 3′-PUA-triazole-histone DPC has two differences. First, the ε-CH 2 of the lysine is replaced by an oxygen atom, which has a minor effect on the size . Second, the latter has an additional triazole moiety.…”