Live cell visualization and quantification of replication proteins at a single-molecule level in natural and damaged environments.
Research overview
DNA replication is one of the most fundamental processes in cellular biology. With every round of cell division, a new, accurate copy of the genome must be synthesized, and this is typically achieved with high reliability by the replication machinery, a protein complex with many components called the replisome. However, there are situations when the replication machinery stall, or even collapse. These situations may stem from inherent cellular processes such as the presence of active proteins on the DNA, or from external agents that damage the DNA. Bacterial systems form excellent model systems for understanding the intricacies of DNA replication.
Our research focuses now on studying the spatial-temporal impact that DNA-damaging drugs and UV radiation have on the different components of the replisome, and on accessory helicases, in live bacterial cells. The fundamental knowledge from comprehending these mechanisms is essential to addressing the biology of cancer-associated processes, where there is extensive genome instability resulting from replication anomalies. The resulting insights into replication dynamics may also provide more specific drug targets for antibiotics discovery.
Approach
We employ an array of different techniques depending on the biological questions that we wish to answer. In the past, we have used in vitro approaches to get direct insight into how individual components of the replication machinery contribute to the efficiency and reliability of DNA replication. High-throughput single-molecule techniques and purified components were used to study for example the termination of bacterial replication (see past projects). Currently, we focus on in vivo approaches to examine the dynamics of replication as it occurs within the complexity of the cell.
The in vivo aspect of these studies is key, because our studies will hence probe the natural cellular context, which includes physiological concentrations and representative interaction dynamics within multi-protein complexes that will complement the data available from in vitro experiments. The fundamental knowledge from comprehending these mechanisms is essential to addressing the biology of cancer-associated processes, where there is extensive genome instability resulting from replication anomalies. The resulting insights into replication dynamics may also provide more specific drug targets for antibiotics discovery.
The bacterium E. coli is our experimental workhorse, involving genetic engineering and molecular biology. We use live cell, single-molecule fluorescence, confocal and super-resolution microscopy approaches to address our research questions. To make this possible, we make use of different fluorescence proteins that can be tagged to the replisome as well as other proteins involved in replication. When necessary, microfluidic devices are used for performing long time-lapse fluorescence microscopy. In these devices, E. coli cells are immobilized in growth channels perpendicular to a main trench through which growth medium is actively pumped.
Open student projects
Live cell imaging: understanding bacterial replication and repair in individual cells
Researchers currently involved
- Belén Solano
- Edo van Veen
- Katinka Ligthart
- Theo van Laar