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 η polymerase, but were bypassed following proteolytic processing.

Glyoxal‐Linked Nucleotides and DNA for Bioconjugations and Crosslinking with Arginine‐Containing Peptides and Proteins

“…We analyzed the conjugates by ESI mass spectrometry and succeeded in acquiring the intact mass of the DNA–histone conjugates 30DNA GE_H2B and 30DNA GT_H2B (Table S13, Figures S70 and 71). We also used in‐gel digestion of the conjugate with nucleases and alkaline phosphatase to cleave the DNA to single nucleotides and trypsin to cleave the protein to peptides [24] . The resulting tryptic peptides were analyzed by nanoLC‐ESI + ‐MS/MS on an Orbitrap Elite mass spectrometer.…”

“…We also used in-gel digestion of the conjugate with nucleases and alkaline phosphatase to cleave the DNA to single nucleotides and trypsin to cleave the protein to peptides. [24] The resulting tryptic peptides were analyzed by nanoLC-ESI + -MS/ MS on an Orbitrap Elite mass spectrometer. The analysis showed fragmentation data of a peptide where a modified arginine is crosslinked to the GE nucleoside (Tables S14 and S15, Figure S72).…”