Defining the molecular link between DNA repair and chromatin remodelling

This study investigates how plants detect, signal and repair DNA damage. DNA is essential for growth and reproduction but is constantly being damaged by highly reactive chemicals present in the cell and environmental factors including carcinogens, UVB and soil pollutants such as heavy metals. A single DNA double strand break is sufficient to cause cell death and DNA therefore needs to be continuously repaired if the organism is to survive. The first steps in repair are detection and signalling of DNA damage. It is important that the cell rapidly recognises and signals the presence of DNA damage to minimise the harmful effects on growth and development of the organism. In this study, to be undertaken at the Faculty of Biological Sciences, University of Leeds, we will establish how DNA double strand breaks are detected and repaired using the model plant Arabidopsis thaliana. We will build on our existing knowledge of DNA damage and repair in plants, including exciting recent results that show that the primary detector of DNA damage, termed the MRN complex, interacts with proteins responsible for the modification of histones (proteins that package DNA and regulate its accessibility). This novel result suggests a mechanism by which the cell can signal DNA damage by modifying histones at the site of the break in the very early stages of DNA damage detection and repair. In this proposal, we will test this hypothesis that histone modification plays a crucial early role in DNA repair. We have isolated mutant plants lacking our newly identified histone modifying proteins, and in this study we will see if DNA breaks are processed differently, in terms of histone modification, the rate of repair and the 'unpacking' of DNA. We will also determine the effects of these mutations on the ability of plants to withstand DNA damage. It is important that we understand these signalling processes in plants, as DNA damage signalling and repair is required to allow growth of crop plants in conditions of environmental stress, and failure of these pathways will result in reduced yields and the accumulation of deleterious mutations in future generations. In view of climate change, it is now important that we understand how plants respond to environmental stresses, which will impact on crop survival and yield. As many of the processes are conserved amongst different organisms, these studies may also have important implications for cancer, aging and gene therapy.

Early detection and rapid signalling of DNA damage is essential for the maintenance of the organism's genome. DNA double strand breaks (DSB) are one of the most serious forms of DNA damage and failure to respond appropriately to these lesions leads to chromosome fragmentation, loss of large amounts of genetic information and often results in cell death. The importance of chromatin modification in the DNA damage response is becoming well established, although the molecular events by which histones are modified specifically at the site of DNA damage remain obscure. Recent studies in my lab have provided the first evidence for the mechanism of histone modifier recruitment in the model plant Arabidopsis thaliana, which suggests that DSB detection and chromatin modification are closely linked in plants. We have identified novel interactions between Arabidopsis histone acetyltransferase enzymes and MRE11, which is one of the first proteins to localise to a DSB. MRE11 is a component of the MRE11-RAD50-NBS1 (MRN) complex with roles in DNA damage detection, repair and signalling in eukaryotes. In this study, we will determine the effects of disrupting histone acetylation on growth and viability of Arabidopsis plants under conditions of genotoxic stress and define the roles of MRE11 and interacting histone acetyltransferases in chromatin accessibility, DNA repair and survival of the organism. Although histone acetylation plays critical roles in the signalling and repair of DNA double strand breaks in eukaryotes the mechanism of acetyltransferase recruitment to the DSB is not known. Our studies will advance our fundamental understanding of histone acetylation in eukaryotic DNA repair mechanisms and define the role and importance of histone acetylation in plant DNA repair in both the molecular and physiological contexts. These results will be informative for the genetic improvement of crops, plant breeding strategies and the development of gene targeting technologies.