As DNA reparation enzymes are essential to preserve genome integrity, understanding the regulation pathway dynamics is crucial. Pyrococcus Abyssi NucS is a recently discovered nuclease acting on branched DNA substrates with free ssDNA extremities, called ﬂaps. The biochemical characterization of NucS has shown a nanomolar aﬃnity for both 5' and 3' ﬂaps with the existence of two DNA binding sites (site I and II) as well as a bidirectional activity characterized by the ability to cleave both 5 'and 3 ' ﬂaps. The mechanisms that are responsible for this activity, particularly its association and dissociation modalities, however, remain poorly understood. In order to probe the dynamics of NucS interactions with its substrate, we set up a total internal reﬂection ﬂuorescence microscope that allows the detection and tracking of single interaction events between ﬂuorescently labeled NucS and DNA substrates tethered to a glass surface. Using Quantum dots, we furthermore developed a new colocalization technique based on multilateration that allows for drift correction and colocalization with an accuracy of 50 nm. In our experiments, we investigated the association and dissociation kinetics of the protein-DNA complex and studied the mechanism that regulates the activity of the NucS at diﬀerent conditions. First, we report a bidirectional and oriented binding on 3' and 5' ﬂaps and reveal that NucS/DNA dissociation follows a mechanism that is not dependent on NucS diﬀusion on ssDNA towards the junction but rather by the NucS/ssDNA energy landscape. We moreover demonstrated a central role of salt concentration in the modulation of NucS/DNA interactions. Second, we show that the dissociation mechanism follows single step process with one rate limiting dissociation constant and conclude that NucS can stochastically unbind its substrate in non cleavage conditions at 23 ◦ C. Thereafter, we studied the association and dissociation kinetics of the NucS-DNA complex at 45 ◦ Cin the presence of the repair cofactor PCNA. We demonstrated that PCNA increases the NucS association rate for 3 'and 5' substrates. This indicates that PCNA acts as a platform that facilitates the recognition of the lesion and the speciﬁc loading of NucS at the junction. In addition, we showed that PCNA destabilizes the NucS-DNA complex, presumably by exerting a force to bend the DNA in order to load the ﬂap extremity into the active site II. In the case of 5' ﬂaps, we have shown that the dissociation follows a two-step mechanism, which is independent of the ﬂap length. We thus proposed a model where NucS binds its substrate directly at the junction thanks to its site I and then threads the free extremity of the ﬂap into site II for cleavage with the help of PCNA that enables substrate bending. In this model, the observed dissociation kinetics may be due to the dissociation of the two independent binding sites. We also showed that the dissociation of NucS on 3' ﬂaps follows a ﬁrst-order dissociation process which might be due to the speed of the second step in the proposed mechanism for 5' ﬂaps. Altogether, our results constitute a notable contribution to the characterization of the DNA-NucS interactionsand to thenucleotide excision repair mechanism more generally. The methods we developed furthermore constitute an original way to probe DNA/protein intramolecular kinetics.
NucS Activity Studies at the Single Molecule Level