Figure 7.

19S enrichment at sites of DSBs requires Ub conjugation and processing. (A) 19S enrichment at sites of DNA damage is promoted by repair E3 ligases. U20S treated with Non-T, RNF8 or BRCA1 siRNAs before exposure to 5 Gy irradiation and staining with the antibodies to γH2AX and the 19S subunit PSMC5. (B) 19S enrichment to chromatin around DSB break site requires RNF8. HeLa expressing oestrogen receptor-IPpo-I (HeLa-IPpo-I) treated with Non-T or RNF8 siRNA before treatment with 4-hydroxytamoxifen (4-OHT) to induce endonuclease translocation into the nucleus. Chromatin immunoprecipitation (ChIP) was performed with the indicated antibodies and PCR adjacent to an IPpo-I cut site amplified. The mean fold increase in PCR product between induced and uninduced cells is shown (three replicates/antibody). (C) 19S enrichment to chromatin around DSB break site requires Ubc13. HeLa-IPpo-I treated with Non-T or Ubc13 siRNA and subject to ChIP as above (three replicates). (D) POH1-JAMM^M colocalises poorly with γH2AX on irradiation. U20S transfected with expression constructs for RFP-POH1 and RFP-POH1-JAMM^M for 48 h before exposure to 5 Gy irradiation, triton extraction and staining with the antibodies to γH2AX. (E) POH1, but not POH1-JAMM^M, enriches chromatin near DNA damage sites. HeLa-IPpo-I I transfected with Flag-POH1 or Flag-POH1-JAMM^M for 24 h, treated ±4-OHT for 16 h. ChIP was performed with anti-Flag antibodies and the mean fold increase in enrichment in PCR product between induced and uninduced cells is shown (three replicates/condition). Inset shows cells similarly transfected with Flag-POH1 and analysed by SDS–PAGE and immunoblot for exogenous POH1 and β-actin loading control.