Biophysics in living cells: details of sliding clamp further unravelled (article in Nature Communications by Charl Moolman and Nynke Dekker (BN Nynke Dekker lab) and others)


(by: TU Delft/Communication TNW)
TU Delft researchers have, in collaboration with their colleagues at the University of Oxford and the McGill University, further unraveled the molecular details of the so-called sliding clamp. The β2 clamp is an essential protein in DNA replication and repair. The research results were published on 18 December in the open access scientific journal Nature Communications.

Theβ2sliding clamp is important for DNA replication and repair
The genetic information contained in DNA is indispensable for the survival of a cell. The DNA is copied (replicated) while a cell is growing. This process must be completed before a cell divides. Furthermore, damaged DNA must be repaired. A collection of proteins ensures that both DNA replication and DNA repair occur without any problems, of which the  so-calledβ2 sliding clamp (β2 clamp) is one component. Little was previously known about the number of β2 clamps involved and their dynamics in live cells.

Number of β2sliding clamps
In the course of their research, the scientists used different methods to identify the number of b2 clamps involved in DNA replication and repair. In all experiments, micron-sized channels (microfluidics) were used to immobilise live E. coli cells. Using quantitative fluorescence microscopy, the number of β2 clamps that is bound to the DNA in the cell during a cell cycle was identified. In order to be able to indirectly visualize a β2 clamp in a DNA-bound state, it was fused with a fluorescent protein (image a). By calibrating the intensity of the fluorescent protein, the researchers succeeded in measuring the amount of clamps on the DNA. This revealed that the number of DNA-bound β2 clamps increases directly after replication starts. Nevertheless, the increase stabilises at a number above forty, resulting in the number of β2 clamps remaining largely constant during the cell cycle (image b). This number of stably bound β2 clamps may possibly form a platform for the binding of the other proteins.

Duration of the β2 sliding clamp bond with the DNA
By using super-resolution microscopy, the researchers also succeeded in directly measuring that a b2 clamp remains DNA-bound during replication. The advanced microscopy technology, known as photo activated fluorescence microscopy (PALM), allows a fluorescent molecule that was previously dark to be “turned on”. On the basis of these measurements, the researchers concluded that a single b2 clamp remains bound to the DNA for a few minutes and is therefore not immediately removed.

Follow-up research
These findings are of great importance to understanding DNA metabolism. Many proteins remain bound to DNA via the β2 clamps, enabling them to perform their different tasks. Future research could be focus on answering the question of which proteins are the dominant users of the DNA-bound β2 clamps.

(a) Individual E. coli cells in microchannels. The yellow foci are the DNA-bound β2 clamps in the individual cells. (b) Quantification of the number of β2 clamps bound to the DNA during replication. An increase in the β2 clamps can clearly be observed after initiation, while a similar decrease can be observed after termination.

Further reading
M. Charl Moolman, Sriram Tiruvadi Krishnan, Jacob W.J. Kerssemakers, Aafke van den Berg, Pawel Tulinski, Martin Depken, Rodrigo Reyes-Lamothe, David J. Sherratt and Nynke H. Dekker. “Slow unloading leads to DNA-bound β2 sliding clamp accumulation in live Escherichia coli cells”. Nature Communications (2014).