Ganji, S. R. | bioRxiv Science

bioRxiv2026

Mechanism of K63-linked polyubiquitin recognition and cleavage by the BRCA1-A complex

Deubiquitylases modulate cellular processes by cleaving monoubiquitin or polyubiquitin chains. The ARISC-RAP80 complex partners with BRCA1-BARD1 to form the BRCA1-A super-complex, which recognizes K63-linked ubiquitin chains at DNA damage sites. ARISC-RAP80 contains multiple ubiquitin-binding sites, yet how these influence recognition and cleavage of K63-polyubiquitylated substrates remains unknown. We discover that a composite three-subunit interface allows ARISC-RAP80 to position K63-linked polyubiquitin chains in its catalytic site. Substrate recognition is further supported by RAP80 and non-catalytic ubiquitin-binding sites that impose a compact conformation to K63-polyubiquitylated substrates. This mechanism exploits the inherent flexibility of long ubiquitin chains and differs considerably from other deubiquitylases. Structure-guided mutagenesis validate ubiquitin chain interactions, and cell-based assays demonstrate a functional role of the observed interfaces in chromatin recruitment. Our findings define mechanisms of polyubiquitin chain decoding and cleavage by ARISC-RAP80, linking ubiquitin reading and erasing functions to BRCA1-A mediated DNA damage responses.

biochemistry↗

bioRxiv2025

Design and Characterisation of Photoactivatable and Lysine Reactive o-Nitrobenzyl Alcohol-Based Crosslinkers

Photoreactive groups are invaluable tools in structural proteomics, offering reagent-free activation and temporal control of protein labelling. However, traditional UV-activatable functional groups often produce unstable intermediates and diverse products, making these chemistries difficult to deploy at scale. In this study, we performed a systematic analysis of ortho-nitrobenzyl alcohol (oNBA) reactivity for integration into novel reagents for chemical crosslinking-mass spectrometry. oNBA photochemistry represents a promising alternative to traditional photoactivatable crosslinkers due to its unique specificity towards lysine residues. Here, we synthesised two molecules comprising oNBA functional groups with different substituents and assessed their labelling efficiency against a model protein. To ensure high labelling yields while maintaining a short irradiation time, we constructed a high power 365 nm irradiation device which improves the efficiency of oNBA photolysis. Our studies identified an amide-substituted probe that labels proteins with high efficiency. We next incorporated this optimised oNBA moiety into a homo-bifunctional crosslinker and a hetero-bifunctional crosslinker in combination with an NHS ester, which both resulted in high yields of crosslinked products. Our findings highlight that optimised oNBA-based reactive groups are viable UV-activated warheads that can deliver high labelling yields and efficient protein crosslinking, unlocking a wealth of potential structural proteomics applications.

biophysics↗