Project A has a biological and a technological component: observation of the earliest steps of DNA unwinding by the replicative helicase CMG, and the use of a hybrid microscope incorporating magnetic tweezers (MT) and total internal reflection fluorescence (TIRF) microscopy, in a DNA supercoiling-based assay.
My initial experiments with CMG in conventional magnetic tweezers revealed an unanticipated but significant problem: the means by which the DNA was tethered to the flow cell surface was unstable in the presence of the CMG proteins. This “tethering instability,” due among other things to the close proximity to the surface of the flowcell, would make the observation of CMG firing in my original experimental design impossible. In response, I took the following decisions:
First, I shifted Project A to a different experimental technique: a combination of optical tweezers with force feedback and scanning confocal fluorescence microscopy. This technique permits an experimental design to detect CMG initiation without being vulnerable to the same “tethering instability” as the magnetic tweezers. It also provided the opportunity to interact with recent data suggesting the existence of an early intermediate in CMG assembly. The resulting publication (in preparation) focuses on the dynamics of intermediate formation and progress to a mature Mcm2-7 double hexamer rather than on DNA topological effects.
Second, I directly addressed the tethering instability by collaborating with a fellow postdoc with significant surface chemistry expertise, with the goal of achieving stable DNA tethering in the original configuration. This surface chemistry sub-project was completed this spring, yielding a protocol that permits stable DNA tethering at the surface. There was insufficient time remaining to implement Project A fully as planned, but preliminary experiments to detect DNA helix opening in magnetic tweezers are planned for fall 2020.
Project B. The tethering instability described above made the detection of CMG firing (and the roles of individual firing factors) impossible in magnetic tweezers. However, I was able to leverage another recent development in the field to study the processive behavior of CMG, independent of its firing, using a different experimental preparation called the isolated CMG system (isoCMG).
The DNA tethers used with isoCMG do not have the same constraints on them as the tethers used in the original experiments, so they could be used with the currently available surface preparation methods. This modified version of Project B is currently in progress, expected to be complete in autumn 2020. Preliminary data indicates that the firing factors Mcm10 and RPA do have a pronounced effect on isoCMG unwinding speed.
Projects C and D. Stall torque measurements require the optimal surface chemistry and reconstituted CMG system discussed in previous sections.
The completion of the surface chemistry sub-project did result in a workable protocol for DNA tethering that is robust against the addition of CMG firing factors, so while it was not feasible to begin projects C and D during the time remaining in the project, preliminary measurement of CMG stall torques in MTT is planned for autumn 2020.
