Biology subjects

Gavard, A. E.

Publications and source records attributed to Gavard, A. E..

2 recordsLinked to original sources

bioRxiv2021

Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition

Artemis (DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in DNA double-strand break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Consistently, Artemis deficiency leads to radiosensitive congenital severe immune deficiency (RS-SCID). Artemis belongs to a structural superfamily of nucleases that contain conserved metallo-{beta}-lactamase (MBL) and {beta}-CASP (CPSF-Artemis-SNM1-Pso2) domains. Here, we present crystal structures of the catalytic domain of wild type and variant forms of Artemis that cause RS-SCID Omenn syndrome. The truncated catalytic domain of the Artemis is a constitutively active enzyme that with similar activity to a phosphorylated full-length protein. Our structures help explain the basis of the predominantly endonucleolytic activity of Artemis, which contrast with the predominantly exonuclease activity of the closely related SNM1A and SNM1B nucleases. The structures also reveal a second metal binding site in its {beta}-CASP domain that is unique to Artemis. By combining our structural data that from a recently reported structure we were able model the interaction of Artemis with DNA substrates. Moreover, co-crystal structures with inhibitors indicate the potential for structure-guided development of inhibitors.

biochemistry↗

bioRxiv2020

Crystal Structure of the Werner's Syndrome Helicase

Werner syndrome helicase (WRN) plays important roles in multiple pathways of DNA repair and the maintenance of genome integrity. Recently, loss of WRN was identified as a strong synthetic lethal interaction for microsatellite instable (MSI) cancers making WRN a promising drug target. Yet, structural information for the helicase domain is lacking, which prevents structure-based design of drug molecules. In this study, we show that ATP binding and hydrolysis in the helicase domain are required for genome integrity and viability of MSI cancer cells. We then determined the crystal structure of an ADP bound form of the WRN helicase core at 2.2 [A] resolution. The structure features an atypical mode of nucleotide binding with extensive contacts formed by motif VI, which in turn defines the relative positioning of the two RecA like domains. The structure features a novel additional {beta}-hairpin in the second RecA and an unusual helical hairpin in the Zn2+ binding domain, and modelling DNA substrates based on existing RecQ DNA complexes suggests roles for these features in the binding of alternative DNA structures. We have further analysed possible interfaces formed from the interactions between the HRDC domain and the helicase core by NMR. Together, this study will facilitate the structure-based design of inhibitors against WRN helicase.

biochemistry↗