P005 Structure and function of a novel endonuclease acting on
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P005 Structure and function of a novel endonuclease acting on
P005 Structure and function of a novel endonuclease acting on branched DNA substrates Christophe Creze1,3, Roxane Lestini2, Joelle Kühn2, Mirjam Czjzek3, Didier Flament1, Hannu Myllykallio2 1 Laboratoire de Microbiologie des Environnements Extrêmes, UMR 6197, IFREMER-UBO-CNRS; 2Laboratory of Optics and Biosciences, Ecole Polytechnique, CNRS UMR7645 - INSERM U696; 3Station Biologique de Roscoff Végétaux Marins et Biomolécules UMR 7139 We have shown that Pyrococcus abyssi PAB2263 [dubbed NucS (nuclease for ss DNA)] is a founding member of a novel endonuclease family that is found in many archaea and some bacteria. Structural determination of P. abyssi NucS revealed a two-domain dumbbell-like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non-catalytic ssDNA-binding domain to regulate the cleavage activity at the catalytic site, thus resulting in the specific cleavage at double-stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3’ and 5’ extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. We also show that P. abyssi NucS strongly interacts with the replication clamp PCNA. In order to understand stoechiometry and configuration of the PCNA-NucS complex, we solved solution structures of NucS and PCNA alone and in complex, using small angle X-ray scattering. These data indicate unexpected conformational flexibility of the NucS protein that may be modulated by interactions with PCNA and/or DNA.