Maintenance of genome integrity and DNA repair
Co-workers: Alasdair Gunn, Anna-Lena Kolb, Peggy Paschke, Alina Rakhimova, George Ronson, Seiji Ura
A critical challenge for cells is to protect and preserve genetic information. As such, a specialised network of pathways known as the DNA damage response (DDR) detects, signals and repairs damaged DNA to restore genome integrity. The importance of these pathways in human health is underscored by the observations that defects in the DDR are associated with chromosomal instability, developmental abnormalities, neurodegeneration, immunodeficiency and increased cancer risk. Our work aims to address the fundamental questions of how DNA damage is signalled to facilitate repair, how this maintains genome integrity, and how its de-regulation contributes to tumorigenesis and other disease states.
Figure 1: Accumulation of the DNA damage signalling kinase ATR (Green) at sites of single stranded DNA (red) generated in nuclei following exposure of cells to ionising radiation
(Click to enlarge)
DNA breaks, either on one or both strands of the DNA helix are particularly cytotoxic varieties of DNA lesion. Our work aims to decipher how these breaks are repaired by the single strand break (SSB) and double strand break (DSB) repair pathways respectively, with specific reference to how ADP-ribosyltransferases (ARTs) regulate these events. ARTs are a family of proteins that recognise DNA breaks, become activated, and transfer one or more ADP-ribose units onto proteins either as monomers or heterogeneous branched chains. ADP-ribosylation regulates DNA repair outcome by modulating chromatin structure in the vicinity of the break and/or by recruiting repair and other factors to DNA lesions through ADP-ribose interaction domains located in the proteins.
Our recent work indicates that different ARTs respond to DNA SSBs and DSBs to regulate alternate repair outcomes by recruiting specific repair factors to DNA damage through PAR-interaction domains. Ongoing research exploits a variety of cellular, biochemical and genetic approaches in the model organism Dictyostelium and human cells to address how this regulation is achieved at the molecular level by: 1) Establishing how different ARTs regulate DNA repair pathway choice through modifying alternate substrates and/or catalysing different modification types in response to SSBs and DSBs 2) Identifying and characterising novel ADP-ribose interacting proteins that function in the DDR. 3) Assessing redundancy between ARTs to regulate compensatory repair mechanisms in the absence of canonical SSB and DSB repair pathways.
Figure 2: Regulation of DNA strand break repair pathways by the ARTs PARP1, PARP2 and PARP3 (Click to enlarge)
Our long-term vision is that this multi-disciplinary approach will provide an increased understanding of how DNA repair is achieved, how defects in these pathways can cause a variety of clinical pathologies, and how this knowledge can be exploited to ameliorate the symptoms of these diseases.
Our work is made possible by support and funding from the BBSRC, Cancer Research UK, MRC and NC3Rs.
Gunn A.R., Banos-Pinero B., Paschke P., Sanchez-Pulido P., Ariza A., Day J., Emrich M., Leys D., Ponting C.P., Ahel I. and Lakin N.D. (2016). The role of ADP-ribosylation in regulating DNA interstrand crosslink repair. J. Cell Sci. 129:3845-3858.
Couto C. A.-M., Hsu D.-W., Teo R., Rakhimova A., Lempidaki S., Pears C.J. and Lakin N.D. (2013). Nonhomologous end-joining promotes resistance to DNA damage in the absence of an ADP-ribosyltransferase that signals DNA single strand breaks. J. Cell Sci. 126:3452-3461.
Couto C. A.-M., Wang H.-Y., Green J.C.A., Kiely R., Siddaway R., Borer C., Pears C.J. and Lakin N.D. (2011). PARP regulates non-homologous end-joining through retention of Ku at double strand breaks. J. Cell Biol. 194:367-375.
Hsu D.-W., Kiely, R., Couto, C. A.-M., Wang, H.-Y., Hudson, J.J.R., Borer, C., Pears, C.J. and Lakin, N.D. (2011). DNA double-strand break repair pathway choice in Dictyostelium. J. Cell Sci. 124:1655-1663.
Medhurst, A.L., Warmerdam, D.O., Akerman, I., Verwayen, E.H., Kanaar, R., Smits, V.A.J. and Lakin, N.D. (2008). ATR and Rad17 collaborate in modulating Rad9 localisation at sites of DNA damage. J. Cell Sci. 121:3933-3940.
Adams, K.E., Medhurst, A.L., Dart, D.A. and Lakin N.D. (2006). Recruitment of ATR to sites of ionising radiation induced DNA damage requires ATM and components of the MRN protein complex. Oncogene. 25:3894-3904.
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