4 / 6
E u r o S c i C o n c o n f e r e n c e o n
Protein, Proteomics and
Computational Biology
Biochemistry & Molecular Biology Journal
ISSN: 2471-8084
D e c e m b e r 0 6 - 0 7 , 2 0 1 8
Am s t e r d a m , N e t h e r l a n d s
Proteomics and Computational Biology 2018
Page 16
P
rimases are single-stranded DNA dependent polymerases that synthesize
RNA/DNA primers during replication. A primase, a DNA polymerase and an
helicase compose the replication machinery of the archaeal plasmid pRN1
1
. The
structure of the archaeal functional primase domain has been solved by X-ray
crystallography
2,3
and it revealed an heteromeric structure with a catalytic prim/
pol domain tethered to a novel helix bundle domain. We investigated the NMR
structure of the functional pRN
1
primase domain in complex with a single-stranded
DNA template containing the GTG motif
4
. We showed that the catalytic prim/pol
domain of this 38 kDa enzyme is not required for template binding. Intermolecular
contacts detected exclusively between the helix bundle domain and the DNA led us
to isolate specifically this structurally independent unit. Our results are compatible
with a conformational switch between a template-bound open state and a closed
active complex
3,5,6
. We used multiple NMR dataset to solve the solution structures
of the helix bundle domain in complex with the single-stranded DNA template
alone and upon cofactors addition. Affinity measurements validated our structural
data demonstrating the importance of residues located in helices 10 and 12 for
the interaction with the GTG motif and confirmed the specificity improvement
observed upon cofactors binding. In association with functional assays, these
novel transient structures bring new perspectives and will help us to characterize
the molecular steps required for priming.
Biography
Julien Boudet received his PhD degree in structural biology and
biophysics from the University of Grenoble (Joseph Fourier
University) in France under the supervision of Prof. Jean-
Pierre Simorre. During his thesis, he learned nuclear magnetic
resonance (NMR) spectroscopy and used this powerful
method to investigate proteins and oligonucleotides structures,
molecularmechanismsunderlyingantibioticresistanceandviral
proteins interactions. After graduating, Julien joined the group
of Prof. Frédéric Allain in ETH Zurich as a postdoctoral research
associate. He focused his investigations on the DNA replication
machinery and, in particular on the primase-mediated catalysis.
He set up innovative computational methods to investigate
challenging biological systems and demonstrated the role of
cofactors in improving the specific template recognition by the
pRN1 primase. He is currently developing computational and
analysis tools to assist therapeutic oligonucleotides design.
boudet.julien@gmail.comCombination of NMR methods to solve
key structures of the pRN1 primase in
complex with its substrates
J. Boudet
1
, J.-C. Devillier
2
, T. Wiegand
3
,
L. Salmon
1
, B. Meier
3
, G. Lipps
2
and F. H.-T. Allain
1
1
Institute of Molecular Biology and Biophysics (ETH Zurich, Switzerland)
2
Institute of Biochemistry and Bioanalytics (University of Applied Sciences of
Northwestern Switzerland, Muttenz, Switzerland)
3
Institute of Physical Chemistry (ETH Zurich, Switzerland)
J. Boudet et al., Biochem Mol biol J Volume:4
DOI: 10.21767/2471-8084-C5-019