Lewis Kay, a senior scientist at the Hospital for Sick Children (SickKids) and University Professor in the University of Toronto's Temerty Faculty of Medicine, has been honoured with a Royal Medal by ...

Title: Assistant Professor – Biological Chemistry Executive Area: Academic Affairs College/School/MBU: College of Natural Sciences Department: Chemistry Work Location: Amherst Schedule: Full Time Work...

Hydrogen bond restraints are critical for NMR structure determination, yet their experimental identification can be challenging for marginally stable structures affording insufficient exchange protection in D2O. As an alternative, we explored the use of NMR between pH 10 and 11, conditions that promote rapid exchange, for identifying backbone amide protons involved in H-bonds. We analyzed ∼750 amide sites distributed across ten proteins with known structures. Survival of amide protons at high pH in standard 2D 1H–15N HSQC spectra for 15N-labeled proteins in H2O, or TOCSY for unlabeled proteins, identifies H-bonds with ∼91% accuracy, exceeding the ∼80% accuracy of traditional H/D exchange experiments in D2O. For two α-helical coiled coils and three globular proteins, we performed alkaline unfolding experiments taking advantage of amide NMR signal attenuation from unstructured segments. Increasing the pH led to a progressive loss of native amide proton NMR signals, revealing an unfolding hierarchy where “foldons” remaining at the highest pH had the most persistent H-bonds under EX1 exchange conditions. The foldons observed at high pH are consistent with partially folded structures previously characterized near neutral pH by native state hydrogen exchange, equilibrium unfolding, and protein fragment studies. For β-sheet proteins, foldons correspond to regions with high inter-residue contact density, whereas in coiled coils they demarcate regions with high α-helical propensity. High-pH NMR experiments provide a sensitive, fast, and broadly applicable approach to map H-bonding. Additionally, they offer the opportunity to explore uncharted protein dynamics and unfolding pathways under basic pH conditions.

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The proliferating cell nuclear antigen (PCNA) sliding clamp is a central component of eukaryotic DNA synthesis. In response to DNA damage, PCNA is mono-ubiquitinated by the RAD6 (E2)-RAD18 (E3) complex. However, the structural basis by which RAD18 engages PCNA to direct mono-ubiquitination has remained poorly defined. Mono-ubiquitinated PCNA can subsequently be extended with K48-linked poly-ubiquitin chains that target PCNA for degradation. Ubiquitin-specific protease 1 (USP1) reverses both mono- and poly-ubiquitination of PCNA; accordingly, USP1 inhibition promotes accumulation of mono-ubiquitinated PCNA at replication forks and reduces total PCNA levels, leading to replication defects that are synthetically lethal with BRCA1 deficiency. Here, we combine computational and structural approaches to identify and characterize a SAP domain PCNA-interacting peptide (PIP) motif within RAD18. We demonstrate that this interaction is required for DNA damage-induced PCNA mono-ubiquitination and for PCNA turnover following USP1 loss. Disruption of the RAD18-PCNA interface suppresses ssDNA gap accumulation and reduces USP1 inhibitor sensitivity in BRCA1-deficient cells. Furthermore, cells adapted to prolonged USP1 inhibition exhibit reduced RAD18 levels, suggesting that deregulation of PCNA mono-ubiquitination represents a biologically relevant resistance mechanism. Together, these findings define a structural interface required for RAD18-dependent PCNA mono-ubiquitination and establish it as a key determinant of USP1-BRCA1 synthetic lethality. ### Competing Interest Statement A.D.D. reports consulting for Bayer AG, Bristol Myers Squibb, EMD Serono, Impact Therapeutics, Tango Therapeutics, Roche Pharma, and Covant, Therapeutics; is an Advisory Board member for Impact Therapeutics; and reports receiving commercial research grants from EMD Serono, Moderna, and Tango Therapeutics The Ludwig Center at Harvard National Institutes of Health, https://ror.org/01cwqze88, R35GM156397

Proceedings of the National Academy of Sciences (PNAS), a peer reviewed journal of the National Academy of Sciences (NAS) - an authoritative source of high-impact, original research that broadly spans...

Hao–Fountain syndrome is a rare neurodevelopmental disorder caused by mutations in the deubiquitinating enzyme Ubiquitin-Specific Protease 7 (USP7)...