Selective DNA Damage Slowed Histone Turnover

Abstract

DNA repair requires remodeling of the histones bound to DNA in eukaryotic cells. The remodeling of histone proteins to allow DNA repair enzymes access to the damaged DNA requires histone post‐translational modifications. After repair is complete, it is likely that the modifications are reversed or the modified histone is replaced by a newly synthesized histone. If histones at DNA damage sites is degraded, the turnover of histone proteins during repair will increase. We used stable isotope‐enriched amino acids to label histone proteins prior to DNA damage. The cellular DNA was selectively damaged using 5‐hydroxymethyl‐2′deoxyuridine (HmdU). After metabolism to its triphosphate and incorporation of HmdU during replication, HmdU is recognized as an oxidized thymine base and DNA repair is initiated. To control for the effect of DNA damage on the replication rate, the DNA of cells was also labeled by adding stable‐isotope enriched uridine to the cell culture media. We used LC‐MS and GC‐MS methods to measure the turnover kinetics of histones and DNA, respectively, during DNA damage and repair. Contrary to our expectation of unchanged or accelerated histone turnover, we observed that the turnover of histone H4 slowed when the DNA was damaged. These results imply that replication‐independent histone turnover is slowed, likely because DNA damage inhibits transcription.