Replication protein A dynamically re-organizes on primer/template junctions to permit DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis.

DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, PCNA, carry out DNA synthesis during lagging strand replication, initiation of leading strand replication, and the major DNA damage repair and tolerance pathways. Pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process involving the major single strand DNA (ssDNA)-binding protein complex, RPA, the processivity sliding clamp loader, RFC, PCNA and pol δ. During this process, the interactions of RPA, RFC and pol δ with a P/T junction all significantly overlap. A burning issue that has yet to be resolved is how these overlapping interactions are accommodated during this process. To address this, we design and utilize novel, ensemble FRET assays that continuously monitor the interactions of RPA, RFC, PCNA and pol δ with DNA as pol δ holoenzymes are assembled and initiate DNA synthesis. Results from the present study reveal that RPA remains engaged with P/T junctions throughout this process and the RPA•DNA complexes dynamically re-organize to allow successive binding of RFC and pol δ. These results have broad implications as they highlight and distinguish the functional consequences of dynamic RPA•DNA interactions in RPA-dependent DNA metabolic processes.

Traffic-Calming Measures and Road Traffic Collisions and Injuries: a Spatiotemporal Analysis.

Traffic-calming measures (TCMs) are physical modifications to the road network aimed at making the roads safer. Although studies have reported reductions in road crashes and injuries tied to the presence of TCMs, they have been criticized for their pre-post designs. This study aims to complement our knowledge of TCMs effectiveness by assessing their impact using a longitudinal design. The implementation of eight TCMs, including curb extensions and speed humps, was evaluated at the intersections and census tract levels in Montreal, Canada from 2012 to 2019. The primary outcome was fatal or serious collisions among all road users. Inference was performed using a Bayesian implementation of Conditional Poisson regression in which random effects were used to account for the spatiotemporal variation in collisions. TCMs were generally implemented on local roads, although most collisions occurred on arterial roads. Overall, there was weak evidence that TCMs were associated with study outcomes. However, subgroup analyses of intersections on local roads suggested a reduction in collision rates due to TCMs (median IRR: 0.31; 95% Credible Interval: 0.12 - 0.86). To improve road safety, effective counterparts of TCMs on arterial roads must be identified and implemented.

Interplay of macromolecular interactions during assembly of human DNA polymerase δ holoenzymes and initiation of DNA synthesis.

In humans, DNA polymerase δ (Pol δ) holoenzymes, comprised of Pol δ and the processivity sliding clamp, proliferating cell nuclear antigen (PCNA), carry out DNA synthesis during lagging strand DNA replication, initiation of leading strand DNA replication, and the major DNA damage repair and tolerance pathways. Pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a coordinated process involving the major single strand DNA-binding protein complex, replication protein A (RPA), the processivity sliding clamp loader, replication factor C (RFC), PCNA, and Pol δ. Each of these factors interact uniquely with a P/T junction and most directly engage one another. Currently, the interplay between these macromolecular interactions is largely unknown. In the present study, novel Förster Resonance Energy Transfer (FRET) assays reveal that dynamic interactions of RPA with a P/T junction during assembly of a Pol δ holoenzyme and initiation of DNA synthesis maintain RPA at a P/T junction and accommodate RFC, PCNA, and Pol δ, maximizing the efficiency of each process. Collectively, these studies significantly advance our understanding of human DNA replication and DNA repair.