Radiation Biology and DNA Repair

1 PhD project offered in the IPP summer call 2019

Scientific Background

A sub-pathway of canonical (c)-NHEJ, termed resection-dependent c-NHEJ, repairs approximately 10-20% of ionising radiation (IR)-induced double-strand breaks (DSBs) in G1 and involves end resection, although to a more limited extent compared with resection during homologous recombination (HR). Since resection without the availability of a homologous template can result in loss of genetic information, this pathway is potentially error-prone. Considering that the majority of somatic cells reside in a quiescent or differentiated G0/G1 state and that some cells have long life spans and large genes, DSBs that occur in active genes could pose a serious threat to the genomic integrity if no high fidelity repair pathway is available. Interestingly, recent studies have uncovered a role for RNA during DSB repair. These studies suggest that the repair of resected DSBs could involve RNA templates to restore missing sequence information, thus potentially providing high fidelity repair in actively transcribed genes in G1 phase.

PhD project proposal: RNA-mediated c-NHEJ of resected DNA double-strand breaks in G1 phase

In this project, we plan to examine the possibility of RNA-templated repair during resection-dependent c-NHEJ in G1. We hypothesise that DSBs undergoing resection in G1 include breaks in transcriptionally active regions, as ongoing transcription could provide the template RNA required. The involvement of RNA templates could implicate that resection-dependent c-NHEJ is less error prone than previously thought and less error prone than resection-independent c-NHEJ. Thus, we aim to investigate DSB repair fidelity in different genomic regions in G1 phase. We will use human fibroblasts and the human DivA (DSB inducible via AsiSI) cell line, and study repair of IR- or restriction enzyme-induced DSBs. While IR induces breaks randomly throughout the nucleus, the DIvA cell system expresses AsiSI, which induces DSBs at 100 annotated sites and allows targeted analysis of DSBs in specific genomic regions. To study if the repair fidelity of resection-dependent c-NHEJ is enhanced or diminished, we will employ NGS technologies. We will elucidate the involvement of RNA templates using immunofluorescence (IF) staining and a variety of biochemical methods. In addition to RNA involvement, we will specifically analyse the role of repair factors associated with resection-dependent c-NHEJ in different genomic regions. The results will shed light on the physiological relevance of resection-dependent c-NHEJ and the possible link to RNA-templated DSB repair in G1.

Publications relevant to the project

[1] Jeggo PA and Löbrich M (2017)  DNA non-homologous end-joining enters the resection arena. Oncotarget 8: 93317-93318.

[2] Löbrich M and Jeggo PA (2017)  A process of resection-dependent nonhomologous end joining involving the goddess Artemis. Trends Biochem Sci 42: 690-701.

[3] Biehs R, Steinlage M, Barton O, Juhász S, Künzel J, Spies J, Shibata A, Jeggo PA and Löbrich M (2017)  DNA double-strand break resection occurs during non-homologous end joining in G1 but is distinct from resection during homologous recombination. Mol Cell 65: 671-684.e5.

[4] Barton O, Naumann SC, Diemer-Biehs R, Künzel J, Steinlage M, Conrad S, Makharashvili N, Wang J, Feng L, Lopez BS, Paull TT, Chen J, Jeggo PA and Löbrich M (2014)  Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1. J Cell Biol 206: 877-894. 

[5] Beucher A, Birraux J, Tchouandong L, Barton O, Shibata A, Conrad S, Goodarzi AA, Krempler A, Jeggo PA and Löbrich M (2009)  ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2. EMBO J 28: 3413-3427.

[6] Riballo E, Kühne M, Rief N, Doherty A, Smith GCM, Recio MJ, Reis C, Dahm K, Fricke A, Krempler A, Parker AR, Jackson SP, Gennery A, Jeggo PA and Löbrich M (2004)  A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to γ-H2AX foci. Mol Cell 16: 715-724.

[7] Goodarzi AA, Noon AT, Deckbar D, Ziv Y, Shiloh Y, Löbrich M and Jeggo PA (2008)  ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin. Mol Cell 31: 167-177..

 

Contact Details

Prof. Markus Löbrich

Technical University of Darmstadt
Biology Department
Radiation Biology and DNA Repair

Schnittspahnstraße 13
64287 Darmstadt

+49 6151 16-24620

Email