E3 ubiquitin-protein ligase required for accumulation of repair proteins to sites of DNA damage. Acts with UBE2N/UBC13 to amplify the RNF8-dependent histone ubiquitination. Recruited to sites of DNA damage at double-strand breaks (DSBs) by binding to ubiquitinated histone H2A and H2AX and amplifies the RNF8-dependent H2A ubiquitination, promoting the formation of 'Lys-63'-linked ubiquitin conjugates. This leads to concentrate ubiquitinated histones H2A and H2AX at DNA lesions to the threshold required for recruitment of TP53BP1 and BRCA1. Also recruited at DNA interstrand cross-links (ICLs) sites and promotes accumulation of 'Lys-63'-linked ubiquitination of histones H2A and H2AX, leading to recruitment of FAAP20/C1orf86 and Fanconi anemia (FA) complex, followed by interstrand cross-link repair. H2A ubiquitination also mediates the ATM-dependent transcriptional silencing at regions flanking DSBs in cis, a mechanism to avoid collision between transcription and repair intermediates. Also involved in class switch recombination in immune system, via its role in regulation of DSBs repair. Following DNA damage, promotes the ubiquitination and degradation of JMJD2A/KDM4A in collaboration with RNF8, leading to unmask H4K20me2 mark and promote the recruitment of TP53BP1 at DNA damage sites. Not able to initiate 'Lys-63'-linked ubiquitination in vitro; possibly due to partial occlusion of the UBE2N/UBC13-binding region. Catalyzes monoubiquitination of 'Lys-13' and 'Lys-15' of nucleosomal histone H2A (H2AK13Ub and H2AK15Ub, respectively).
Background References
1. Wei M et al. SENP1 Decreases RNF168 Phase Separation to Promote DNA Damage Repair and Drug Resistance in Colon Cancer. Cancer Res. 2023 Sep
2. Qiu L et al. The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair. Nucleic Acids Res. 2023 Sep
Western blot analysis of RNF168 on different lysates with Rabbit anti-RNF168 antibody (HA722825) at 1/1,000 dilution.
Lane 1: K-562 cell lysate Lane 2: A431 cell lysate Lane 3: MDA-MB-231 cell lysate Lane 4: SK-Br-3 cell lysate Lane 5: HeLa cell lysate Lane 6: 293T cell lysate Lane 7: HepG2 cell lysate Lane 8: U-2 OS cell lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 65 kDa Observed band size: 75 kDa
Exposure time: 3 minutes; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722825) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
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