Periodic Reporting for period 1 - TOPOREF (The Effect of DNA Topology on Eukaryotic Replication)
Periodo di rendicontazione: 2018-06-01 al 2020-05-31
The replisome is the protein complex that carries out DNA replication. In eukaryotes, the core of the replisome is a helicase called CMG that unwinds the DNA double helix. Replication errors are a factor in the development of cancer. New insights into eukaryotic replication could contribute to novel treatments or diagnostic tools for cancer that would be of great social and economic value.
Over the past 30 years, our knowledge of the yeast replisome has grown to encompass all its protein components and a growing list of factors that mediate its interaction with chromatin. The order of assembly of the replisome is well understood, as are the mechanisms by which it is kept in sync with the cell cycle. However, the physical processes that activate the replisome and the dynamics of its interaction with the DNA substrate remain under active investigation.
On a higher level, every aspect of replication hinges on the physical manipulation of DNA. Accordingly, replication must involve the controlled application, monitoring, and relaxation of torque on that molecule. In this proposal, I aim to address the relationship between DNA torque and twist during replication.
Project A concerns the assembly of the replisome. At what point in CMG assembly does melting of the DNA double helix occur? What are the accompanying timescales and roles of critical factors? What is the effect of supercoiling and tension in the DNA?
Project B picks up the kinetics of the advancing replisome. My goal is to characterize how the rate and pause dynamics of the replisome depend on torque on the DNA as well as on critical firing factors.
Project C concerns measurement of the torque required to stall the replisome. If the replisome advances far enough in the absence of certain accessory factors, the torque that builds up in front of the replisome will eventually stall the replicative helicase when it can no longer generate the force needed to melt the strands.
Project D. The basic subunit of chromatin is the wrapping of DNA around nucleosomes. The presence of nucleosomes decreases the torsional stiffness of DNA, suggesting that they are able to absorb torque generated by the replisome by flipping their orientation. By comparing the stalling dynamics of replisomes in the presence and absence of nucleosomes, I will determine whether nucleosomes allow the replisome to advance further before stalling.
Over the past 30 years, our knowledge of the yeast replisome has grown to encompass all its protein components and a growing list of factors that mediate its interaction with chromatin. The order of assembly of the replisome is well understood, as are the mechanisms by which it is kept in sync with the cell cycle. However, the physical processes that activate the replisome and the dynamics of its interaction with the DNA substrate remain under active investigation.
On a higher level, every aspect of replication hinges on the physical manipulation of DNA. Accordingly, replication must involve the controlled application, monitoring, and relaxation of torque on that molecule. In this proposal, I aim to address the relationship between DNA torque and twist during replication.
Project A concerns the assembly of the replisome. At what point in CMG assembly does melting of the DNA double helix occur? What are the accompanying timescales and roles of critical factors? What is the effect of supercoiling and tension in the DNA?
Project B picks up the kinetics of the advancing replisome. My goal is to characterize how the rate and pause dynamics of the replisome depend on torque on the DNA as well as on critical firing factors.
Project C concerns measurement of the torque required to stall the replisome. If the replisome advances far enough in the absence of certain accessory factors, the torque that builds up in front of the replisome will eventually stall the replicative helicase when it can no longer generate the force needed to melt the strands.
Project D. The basic subunit of chromatin is the wrapping of DNA around nucleosomes. The presence of nucleosomes decreases the torsional stiffness of DNA, suggesting that they are able to absorb torque generated by the replisome by flipping their orientation. By comparing the stalling dynamics of replisomes in the presence and absence of nucleosomes, I will determine whether nucleosomes allow the replisome to advance further before stalling.
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.
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.
Comparatively few single-molecule experiments have been done with the reconstituted CMG replisome, so a validated assay for CMG loading in optical tweezers (publication in progress) is of general interest to the field. To analyse this data, I have coauthored new software with my group’s IT technician, which will be generally useful to researchers using the same or similar optical trapping / confocal fluorescence instruments.
The optimized surface preparation developed to solve the DNA tether instability will be of interest for single-molecule experimentalists using a wider array of biological systems, as the problems of stable DNA tethering are both general and acute for systems of increased biological complexity.
During the fellowship, I have presented my work widely at international and national (Dutch) conferences with a variety of topics, ranging from the technique-oriented single-molecule biophysics conferences to biology-oriented meetings on DNA replication and repair.
I have also contributed extensively to teaching at my host institution by supervising a Masters student project, contributing to the training of two new PhD students, conducting undergraduate lectures on magnetic tweezers, and tutoring an undergraduate single-molecule microscopy lab.
Finally, I have been actively developing novel ways to carry out public engagement activities while under Covid-19 restrictions. I produced a series of video lectures for undergraduates for the spring 2020 nanobiology course at my host institution, and I am developing two short, interactive video introductions to single-molecule biophysics and DNA replication, to stimulate discussion and interest in science for a target audience of high school students.
Note: no website has been developed for this project.
The optimized surface preparation developed to solve the DNA tether instability will be of interest for single-molecule experimentalists using a wider array of biological systems, as the problems of stable DNA tethering are both general and acute for systems of increased biological complexity.
During the fellowship, I have presented my work widely at international and national (Dutch) conferences with a variety of topics, ranging from the technique-oriented single-molecule biophysics conferences to biology-oriented meetings on DNA replication and repair.
I have also contributed extensively to teaching at my host institution by supervising a Masters student project, contributing to the training of two new PhD students, conducting undergraduate lectures on magnetic tweezers, and tutoring an undergraduate single-molecule microscopy lab.
Finally, I have been actively developing novel ways to carry out public engagement activities while under Covid-19 restrictions. I produced a series of video lectures for undergraduates for the spring 2020 nanobiology course at my host institution, and I am developing two short, interactive video introductions to single-molecule biophysics and DNA replication, to stimulate discussion and interest in science for a target audience of high school students.
Note: no website has been developed for this project.