Our goal is to understand how DNA is organized under confinement, and what are the consequences of this confinement on the functional properties of the genetic material.
Our hope is: <br />1) to get experimental evidence of multiple types of DNA–DNA correlations and to identify the parameters that can control them,? <br />2) to understand how the confinement of the DNA chain interferes with these correlations and estimate the weight of each of the critical parameters involved,? <br />3) to complete the structure of the DNA crystal inside the full capsid by combining different structural approaches. <br />
We will develop CryoTEM and XRay approaches.
Too early- We are just starting the project
We guess that?
- a better understanding of DNA-DNA correlations may be essential if, as suggested by some authors (but not proved yet), such correlations may help specific DNA sequences recognize each other in a complex crowded environment. Our approaches may open new routes in the field.
- a better understanding of phage infection processes may be useful to progress in strategies of “phage therapy”, an approach based on the direct application of phages on the infected area.
This project which includes three teams of physicists and biologists (F. Livolant, LPS Orsay; D. Durand, IBBMC Orsay and D. Chretien, TIPs Rennes), aims to elucidate the interactions and correlations between DNA helices maintained at very close interdistance. The incidence of such correlations may be essential in terms of biological functionality. The objectives are to understand these interactions in three different conformations (bundles, toroids and related shapes and globules). Bacteriophages (viruses that infects bacteria) present the advantage of maintaining in their protein capsid a long chain of DNA at concentrations that cannot be achieved in vitro. By inducing a controlled partial ejection of the genome, we can perform experiments on individual DNA chains, confined in the volume of the capsid itself. Based on the experience of some partners with bacteriophage T5, our objectives are:
1. To study the interactions between DNA. We plan:
- To identify the interactions and possible correlations between DNA chains by varying several experimental parameters (ionic conditions, osmotic stress). We will also test the effect of chain length (50 nm to several microns). The methods of X-ray diffraction and cryo-electron microscopy will be used to complement one another in order to get the best resolution.
- To elucidate the crystal structure of DNA in the volume of the full capsid by cryo-electron microscopy and tomography of individual particles. This will be complemented by X-ray diffraction experiments on solutions of phages. This study will need to overcome the resolutions currently available by cryo-electron tomography. Capsids of different sizes will be used to vary the confinement stress and curvature of the chains.
- To elucidate by the same methods the structure of the toroids and related forms of DNA under confinement (intracapsid) and to identify deviations from the canonical helical structure of DNA.
2. To consider the phages in their natural context (interacting with their host bacteria) to identify the native conformation of the DNA in the capsid i) during the ejection of the phage genome into the host bacterium and ii) during the packaging of DNA into newly formed capsids. The DNA/DNA and DNA/capsid interactions will be analysed and we will test the hypothesis of non symmetry of the ejection and packaging processes.
The consortium brings together all skills required for the preparation of the biological material and for structural analyses by cryo-electron microscopy and cryo-tomography and by Xray diffraction.
Madame LIVOLANT Françoise (Laboratoire public)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IBBMC Institut de Biochimie et Biophysique Moléculaire et Cellulaire
TIPS UMR6290 IGDR Institut de Génétique et Développement de Rennes
Help of the ANR 339,976 euros
Beginning and duration of the scientific project: January 2013 - 36 Months