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The mean dsb induction frequency of all 16 cell lines was 8.1 × 10−12 dsb/Gy/Da.
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Consistent with previously published results for γ-H2A.X around a DSB, we observed a rapid accumulation of γ-H2A.X within an hour of DSB induction.
Therefore, the objective of this study was to investigate DSB induction by Cr VI) exposure with the overarching hypothesis that S phase-dependent DSBs are produced by Cr VI) exposure.
Immunoblots showing the levels of (a) H3K36me3 and (b) H3K36me2 after DSB induction by phleomycin.
H3K36me3 was increased, although modestly, within 30 min of galactose-induced DSB and tapered off ~75 min after DSB induction (Fig. 4c).
We observed that even within 30 min after DSB induction, there is more RPA around a DSB in a set2Δ as compared with WT (Supplementary Fig. 6a,b).
DSB induction and γ-H2AX immunostaining were performed using an OxiSelectTM DNA DSB Staining Kit (CELL BIOLABS, INC ., according to the manufacturer's protocol.
Interestingly, a positive correlation between the extent of histone acetylation and DSB formation is evident in human cultured cells, suggesting that the mechanism of DSB induction is also valid in animals.
Chromatin immunoprecipitation (ChIP) analysis revealed that H3K36me3 levels increase in a Set2-dependent manner following HO-induced DSB induction (Fig. 2e).
Additionally, after 30 min of DSB induction, we can see significant reduction in the level of RNAPII (Supplementary Fig. 4g), a result consistent with our global analysis.
See also Supplementary Fig. 5. (e) DSB induction results in a Set2-dependent increase in histone H3K36me3 levels proximal to the HO break.
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