DNA damage responses in mammalian cells and their contribution to human health disorders; the end-stage.

All cells in our body carry the same genetic information encoded within the DNA sequence. The entire DNA sequence represents our genome. Our DNA is constantly damaged by exogenous and endogenously arising reactive chemicals. It is important that our genomes are stably maintained without any sequence changes. The maintenance of genomic stability is crucial for cancer avoidance and for normal growth and development. Genomic stability is maintained by DNA damage response mechanisms. Individuals impaired in such mechanisms have been described and can display elevated cancer predisposition, abnormal development and/or enhanced sensitivity to environmental agents such as ionizing radiation (IR). My laboratory studies the response to DNA double strand breaks (DSBs), which arise when both strands of the DNA molecule are broken in close proximity. DSBs are commonly induced by exposure to IR. The major process that repairs DSBs is called DNA non-homologous end-joining (NHEJ). The MRC has funded my laboratory for more than ten years to study the response to DSBs. The aim of this proposal is to complete the ongoing programme of work prior to my retirement. In one component of work, we have shown that the organization of DNA influences how it is repaired. DNA regions that are rarely used are more highly compacted than those that encode proteins that are required frequently. The most highly compacted DNA, called heterochromatin, is repaired more slowly than more open DNA and the repair requires additional proteins. We will seek insight into this process.
The development of the immune response requires the creation of a diverse repertoire of cells that recognize different antigens. This is achieved by the creation of DSBs which are rejoined inaccurately. Curiously cells utilize NHEJ to create diversity during immune development and to maintain stability after accidental DSB formation. Patients defective in NHEJ proteins have been described and show immunodeficiency as well as IR sensitivity. They also frequently have small heads at birth demonstrating that NHEJ is required for normal neuronal development. We have characterised a mouse model for one such disorder called LIG4 Syndrome. We will complete the analysis of LIG4 mice to gain insight into the basis underlying the small head phenotype of LIG4 patients. Finally, we have also examined the basis underlying Seckel Syndrome, another disorder conferring small heads and developmental delay. We have identified genetic defects for Seckel Syndrome and will complete our current studies on the identified proteins.

DNA non-homologous end-joining (NHEJ) is the major process that repairs DNA double strand breaks (DSB) that arise during immune development and following exposure to ionising radiation. The kinase, ATM, lies at the centre of a signal transduction response to DSBs. The Jeggo laboratory has studied the DNA damage response pathways responding to DSBs for the past ten years. This application is to pursue the ongoing work to retirement. Previous studies have shown that although NHEJ occurs largely independently of ATM signalling, a component of DSB repair requires ATM and the nuclease, Artemis. DSBs that require ATM and Artemis are located at regions of heterochromatic DNA, demonstrating that chromatin structure significantly affects DSB repair. One aim is to examine the impact of heterochromatin on DSB repair and damage response signalling and to further our understanding of the roles of ATM and Artemis. The cell cycle phase also influences how heterochromatic DSBs are repaired. In G1 phase, they are repaired by a process that requires DNA ligase IV, an NHEJ protein but in G2 phase, they are repaired by homologous recombination. We will examine factors influencing the distinct processes in G1 versus G2 phase. LIG4 Syndrome, which is characterised by immunodeficiency and microcephaly, represents a human disorder conferred by hypomorphic mutations in DNA ligase IV, an essential NHEJ protein. We have characterised a mouse model for LIG4 Syndrome and will exploit this model system to examine the impact of NHEJ during development. We will complete an examination of the role of DN A ligase IV during embryonic neuronal development, providing insight into the basis underlying the microcephaly observed in LIG4 patients and also examine the intestinal crypt. These systems allow an examination of damage responses functioning in stem cells in vivo. Finally, we have identified genetic defects in Seckel Syndrome, a human disorder conferring microcephaly and developmental delay. We will complete ongoing studies on recently identified causal genes for SS. The aim is to complete ongoing work in high quality publications, to complete the training of personnel employed on the grant and to promote the exploitation of our findings by colleagues.