Objective
In eukaryotic cells the DNA is packaged into nucleosomes and higher order structures which lead to a condensation and protection of the DNA. During important cellular processes such as transcription or replication access to the DNA has to be granted. This is facilitated by ATP dependent nucleosome remodelling. The mechanistic details how the involved remodelling complexes succeed in providing access to the nucleosomal DNA are currently in spite of large experimental efforts not well understood. The aim of the proposal is to unravel the molecular mechanism of nucleosome remodelling using single-molecule fluorescence resonance energy transfer (FRET). By putting labels on the nucleosomal DNA, the histones or the remodellers we will - step by step - determine the conformational changes that occur during remodelling and use this information to build a mechanistic model. We will also use the controlled assembly of 30 nm fibers from purified components in order to determine how remodelling occurs in this structurally restricted environment. The large size of the chromatin fibers will dictate that in addition to FRET measurements, which will again be used to investigate local motion, super-resolution microscopy need to be employed to obtain information about long-distance movements. To this end we will use stochastic optical reconstruction microscopy (STORM), which allows for accuracy below 10 nm, thus ideally complementing the FRET approach. In summary our experiments will lead us to a mechanistic understanding of ATP dependent nucleosome remodelling, both on mono-nucleosomes as well as in higher order structures. This knowledge will in return stimulate new initiatives aimed at understanding the nature and regulation of chromatin dynamics in vivo.
Fields of science
Keywords
Call for proposal
ERC-2009-StG
See other projects for this call
Funding Scheme
ERC-SG - ERC Starting GrantHost institution
89081 Ulm
Germany