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.