Community Research and Development Information Service - CORDIS


DynGenome Report Summary

Project ID: 312221
Funded under: FP7-IDEAS-ERC
Country: Netherlands

Periodic Report Summary 2 - DYNGENOME (The Dynamics of Genome Processing)

We have advanced our understanding of, helicases, polymerases, and other molecular machines that help to read out and copy our genome. We have addressed three different phases of functioning for these machines: initiation, elongation, and termination. For example, we have learned about the loading behavior of a key component of the T7 replisome, its helicase, onto DNA during initiation. We find that this hexameric helicase can form around the DNA itself, as opposed to necessarily pre-forming in solution. This provides flexibility in the initiation of replication, and is of general interest because similar types of helicases are used in many different organisms . We have also investigated the activity of RNA polymerases as they transcribe RNA from DNA. Here, we have focused on the analysis of the pauses made by RNA polymerases during elongation. This sheds light on the mechano-chemical steps that these nanometer-sized enzymes make. Lastly, we have completed studies on the Tus protein involved in E. coli replication termination. This has allowed us to determine how this protein can form a protein-DNA lock that halts replisomes.

As replisomes normally function on DNA covered with DNA-binding proteins such as transcription factors, other molecules machines, or, in the case of eukaryotic systems, nucleosomes, we have examined are a primary candidate, we have also investigated the compaction of DNA by nucleosomes and tetrasomes. This has shown us that tetrasomes in particular exhibit strong conformational flexibility on DNA, possibly making it easier for molecular machines to advance along chromatin.

In parallel with these biophysical studies, we have completed several methodological developments on magnetic tweezers. We have performed in-depth studies of the properties of magnetic beads and on the calibration of magnetic tweezers. We have also advanced the development of high-speed and high-throughput magnetic tweezers suitable for studying the E. coli replisome. We have started to use these more advanced experimental setups for the first high-throughput studies of molecular machines. Our development of angular tracking technologies is in use on the above-mentioned projects on chromatin but we have also demonstrated their utility for structural studies of RNA.

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