Temperature dependent γH2AX binding to DNA

Abstract

γ-H2AX, the serine 139 phosphorylated form of histone H2AX, is one of the earliest DNA double-strand breaks repair responses. Therefore, the quantity of γ-H2AX-bound DNA usually reflects the extent of DNA damage and consequently, mutagenic potential. The purpose of this study was to assess the cellular evidence regarding whether and how temperature is related to mutation. This study applies our novel assay technique to measure the quantity of γ-H2AX-bound DNA by PCR. Chromatin immunoprecipitation technique was applied to select for the γ-H2AX-bound DNA complex. Then, the amount of precipitated DNA was measured by long interspersed element-1 (LINE-1) repetitive sequences realtime PCR. We observed a positive correlation between the amount of precipitated LINE-1 sequences and the exposure to temperature. The quantity of γ-H2AX-bound DNA increased significantly when HeLa was incubated in 47 °C for 30 min. In contrast, incubation at 4 °C for 30 min reduced the γ-H2AX-bound DNA quantity. Precise quantification of γ-H2AX-bound DNA will be a tool to prove whether there is a linkage between temperature and mutagenic effect.

Replication independent DNA double-strand break retention may prevent genomic instability

“…Therefore, we investigated whether the relationship between RIND-EDSBs and γ-H2AX is reversed under our conditions. γ-H2AX-bound DNA was obtained by Chromatin Immunoprecipitation (ChIP) [ 60 ] using a γ-H2AX antibody, and bound LINE-1 sequences were quantified by real-time PCR using 5' LINE-1 primers [ 24 ] (Additional file 4 and 5 ). LINE-1s near RIND-EDSBs were consistently hypermethylated [ 1 ].…”

“…The ChIP assay was performed essentially as previously described with some modifications [ 24 , 60 ]. The chromatin fragments were immunoprecipitated with anti-phospho-Histone H2AX monoclonal antibody (Upstate, Charlottesville, VA, USA) or normal mouse IgG antibody as a negative control (Santa Cruz Biotechnology).…”