Secondary Interaction Interfaces with PCNA Control Conformational Switching of DNA Polymerase PolB from Polymerization to Editing

Replicative DNA polymerases
(Pols) frequently possess two distinct
DNA processing activities: DNA synthesis (polymerization) and proofreading
(3′–5′ exonuclease activity). The polymerase
and exonuclease reactions are performed alternately and are spatially
separated in different protein domains. Thus, the growing DNA primer
terminus has to undergo dynamic conformational switching between two
distinct functional sites on the polymerase. Furthermore, the transition
from polymerization (pol) mode to exonuclease (exo) mode must occur
in the context of a DNA Pol holoenzyme, wherein the polymerase is
physically associated with processivity factor proliferating cell nuclear antigen (PCNA) and primer–template
DNA. The mechanism of this conformational switching and the role that
PCNA plays in it have remained obscure, largely due to the dynamic
nature of ternary Pol/PCNA/DNA assemblies. Here, we present computational
models of ternary assemblies for archaeal polymerase PolB. We have
combined all available structural information for the binary complexes
with electron microscopy data and have refined atomistic models for
ternary PolB/PCNA/DNA assemblies in pol and exo modes using molecular
dynamics simulations. In addition to the canonical PIP-box/interdomain
connector loop (IDCL) interface of PolB with PCNA, contact analysis
of the simulation trajectories revealed new secondary binding interfaces,
distinct between the pol and exo states. Using targeted molecular
dynamics, we explored the conformational transition from pol to exo
mode. We identified a hinge region between the thumb and palm domains
of PolB that is critical for conformational switching. With the thumb
domain anchored onto the PCNA surface, the neighboring palm domain
executed rotational motion around the hinge, bringing the core of
PolB down toward PCNA to form a new interface with the clamp. A helix
from PolB containing a patch of arginine residues was involved in
the binding, locking the complex in the exo mode conformation. Together,
these results provide a structural view of how the transition between
the pol and exo states of PolB is coordinated through PCNA to achieve
efficient proofreading.