Structure and function of the nucleus in DNA damage signaling and repair

A fundamental property of all living organisms from bacteria to humans, is the ability to correct genetic errors by repairing DNA. In multicellular organisms, DNA is found in the nucleus packaged with histone proteins to form chromatin. Damage to DNA is more difficult to find and repair when it is packaged into chromatin. However, one of the histone proteins in chromatin becomes phosphorylated (termed gamma-H2A.X), marking that chromatin for DNA repair and producing punctate spots or foci that can be seen with a light microscope. These "repair foci" are believed to act as beckons for the recruitment of enzymes that repair DNA. Repair foci are sometimes associated with particular sub-regions or domains of the nucleus such as the promyelocytic leukemia nuclear body (PML NB) and the nuclear lamina. Although repair foci have been immensely useful in the identification of novel DNA repair factors by virtue of their co-localisation with gamma-H2A.X, we know virtually nothing about their ultrastructure or how changes in their structure or location in the nucleus relate to the process of DNA repair. We have developed a high resolution electron microscopy technique, termed correlative light and electron spectroscopic imaging (LM/ESI) that allows the ultrastructure of chromatin to be visualised at DNA repair foci within mammalian cells. In this proposal, we will study DNA repair at a single DNA double-strand break (DSB) induced by the mega-endonuclease I-SceI in mammalian cells. Using LM/ESI in combination with live-cell microscopy we will correlate changes in the recruitment and spatial organization of DNA repair factors with changes in the structure and post-translational modification of chromatin at a single DSB over time. This approach will enable the inter-relationship between chromatin structure during DNA repair and the location of DNA breaks, with respect to subnuclear domains, to be characterised for the first time. These studies will greatly advance our understanding of how nuclear structure and chromatin organization contribute to the fundamental process of DNA repair in vivo.