DNA replication requires the coordination of numerous proteins that assemble to form the replisome. In many bacteria, more than one DNA polymerase functions as part of the replisome to complete chromosome and plasmid replication. Further, during DNA replication DNA polymerases can stall or encounter damage in the template strand. Therefore, in our study, we asked how two DNA polymerases are coordinated at a replisome in a living cell. We also asked how a DNA replication block or DNA damage affects the dynamic recruitment of DNA polymerases to the replisome.
The cover image for the February 19th issue of the Biophysical Journal combines superresolution imaging and model replication fork structures to depict the dynamic and cooperative process of DNA replication in Bacillus subtilis. Our illustration shows DNA polymerase tracking during normal replication and following replication fork stress using damage-independent and damage-dependent conditions. Two replicative DNA polymerases, PolC and DnaE, and a processivity clamp loader subunit, DnaX, show complementary fast, moderate, and dwelling behavior. During normal replication, we show (top) that PolC and DnaX show dwelling behavior at the replication fork, while DnaE, a DNA polymerase responsible for the synthesis of DNA from an RNA primer, is untrackable. When DNA synthesis by PolC is arrested using a chemical inhibitor (HPUra) that binds to the active site (middle: HPUra represented by the bolt), we observe longer dwell times for PolC and DnaX at the fork, while DnaE still remains untrackable. When we add a DNA crosslinking agent, mitomycin C (bottom: MMC represented by an exaggerated chemical crosslink of the parental DNA strands), the cross-linked strands would prevent further DNA duplex unwinding and arrest DNA synthesis. During this condition, we observed long dwelling behavior at the replication fork for the clamp loader protein DnaX, while PolC becomes more mobile and DnaE remains difficult to track, indicating a large mobile population of molecules. The lack of dwelling behavior for DnaE following DNA damage is important because expression of DnaE is known to be upregulated following challenge with MMC.
This cover illustration was motivated by our work on DNA replication and repair machines. In this study, we have used single-molecule tracking in combination with computation, biochemistry, and other experimental approaches to understand how genome integrity is maintained during DNA replication and repair. We then use the dynamical information to build conceptual models for how protein exchange occurs during a replication or repair event. This study is important to the field because it provides a real-time, high-resolution approach to the protein dynamics that occur during normal replication and following damage-dependent and damage-independent fork arrest.
-Lyle Simmons and Julie Biteen