Matteo De March & Alfredo De Biasio

(2017): The dark side of the ring: role of

the DNA sliding surface of PCNA, Critical
Reviews in Biochemistry and Molecular
Biology, DOI:
10.1080/10409238.2017.1364218
Objective:
Determine the molecular mechanism of clamp sliding.
Structural basis of
human PCNA sliding on
DNA
MATTEO DE MARCH, NEKANE MERINO, SUSANA BARRERA -
VILARMAU, RAMON CREHUET, SILVIA ONESTI, FRANCISCO J.
BLANCO, & ALFREDO DE BIASIO
Diagram of
Methods

Matteo De March, Nekane

Merino, Susana Barrera-Vilarmau,
Ramon Crehuet, Silvia Onesti,
Francisco J. Blanco, & Alfredo De
Biasio
Results
Structure of the PCNADNA complex
Dynamics of the PCNADNA interaction
Structure of the PCNA
DNA complex.
X Ray Crystallography
DNA in the clamp channel forms a 15 angle
with the three-fold rotation axis of the
clamp.
The crystallographic interface is composed
of six conserved basic residues distributed
on five a-helices, across two subunits,
establishing polar contacts with five
consecutive phosphates of a single DNA
strand
Supplementary Figure 1
NMR Results
Titration of PCNA with dsDNA shows weak
binding (KDB0.7mM -Dissociation constant)
to the inner side of the ring.
Single stranded portion of a primed DNA
substrate did not interact with the protein
binding (PIP-box) pocket of PCNA,
suggesting that bacterial and eukaryotic
clamps recognize primed DNA differently.
Dynamics of the PCNA
DNA interaction
Molecular Dynamics
DNA migrates from the crystallographic position to
an alternative position, and subsequently to a
central location with minimal interactions with the
protein, to eventually collapse into a stable state.
DNA duplex longer than 10bp will simultaneously
bind two sets of B-helix matching residues on two
PCNA subunits in a dynamic way.
MD simulations show that many of the PCNA
interfacial residues can randomly switch between
adjacent DNA phosphates on a sub-nanosecond
time scale
MD simulation 1
Molecular Dynamics
DNA migrates from the crystallographic position to
an alternative position, and subsequently to a
central location with minimal interactions with the
protein, to eventually collapse into a stable state.
DNA duplex longer than 10bp will simultaneously
bind two sets of B-helix matching residues on two
PCNA subunits in a dynamic way.
MD simulations show that many of the PCNA
interfacial residues can randomly switch between
adjacent DNA phosphates on a sub-nanosecond
time scale
MD simulations 2
Molecular Dynamics
DNA migrates from the crystallographic position to
an alternative position, and subsequently to a
central location with minimal interactions with the
protein, to eventually collapse into a stable state.
DNA duplex longer than 10bp will simultaneously
bind two sets of B-helix matching residues on two
PCNA subunits in a dynamic way.
MD simulations show that many of the PCNA
interfacial residues can randomly switch between
adjacent DNA phosphates on a sub-nanosecond
time scale
Discussion and Conclusion
The data supports a molecular sliding mechanism that keeps the
orientation of the clamp invariant relative to the DNA backbone.
Interaction of PCNA with primed DNA that reveal substantial
differences compared to the bacterial system.
K20, K77, K80, R149 and K217 residues are critical for PCNADNA
recognition and for orienting the clamp on DNA.
Proposed mechanism for PCNA sliding would ensure that the
incoming polymerase encounters PCNA in the correct orientation to
efciently restart synthesis.
K14, K20, K80 and K217 residues take part in the binding of human
PCNA to DNA, may play a role in clamp loading by guiding DNA
through the open clamp and into the clamp loader to form a tight
complex.
Discussion and Conclusion
The presence of a dynamic interface, where
the backbone atoms of DNA forms short-
lived interactions with a set of basic residues
facing the internal wall of the protein ring
supports a molecular sliding mechanism that
keeps the orientation of the clamp invariant
relative to the DNA backbone.