McKinnon: DNA Strand Break Repair
Our initial studies identified a differential utilization of each of the two types of double strand break repair pathways in different regions of the developing nervous system [PNAS 103: 10017-22, 2006]. We showed that homologous recombination (HR) function was confined to proliferating cells, while immature postmitotic differentiating cells preferentially utilized non-homologous end-joining (NHEJ). We further established that ATM was uniquely required for DNA damage-induced apoptosis in only the differentiating cells after DNA DSBs where NHEJ, but not HR, occurs. These data have been important for understanding the specific repair pathways required to prevent accumulation of DNA DSBs in the nervous system.
Figure 2. Selective utilization of DNA double strand break repair in the developing nervous system. Germline deletion of DNA double strand break repair factors, DNA ligase IV (Lig4) and Xrcc2 required for homologous recombination (HR) and non-homologous end-joining (NHEJ) respectively, indicates that HR is required for repair in proliferating cells, while NHEJ is mainly involved in DNA repair in differentiating cells. Unrepaired DNA damage activates apoptosis (TUNEL staining, green). Ki67 staining (red) identifies proliferating cells. VZ is ventricular zone and SVZ is sub-ventricular zone.
Figure 3. Utilization of DNA strand break repair pathways during nervous system development. The development of the nervous system occurs via cycles proliferation, differentiation/migration and maturation. These reiterative processes occur as the nervous system develops, and specific DNA repair pathways are important during each of these phases. Three main pathways repair DNA strand breaks. DNA double strand breaks (DSBs) are repaired by either non-homologous end‑joining (NHEJ) or homologous recombination (HR) repair. The dashed line representing NHEJ during proliferation indicates that NHEJ may not be important during DSB repair by HR in the proliferative zone of the nervous system. A different pathway repairs DNA single strand breaks (DSBs), and is operative throughout nervous system development and in the mature nervous system. In some cases, HR can repair DNA single strand breaks during replication (dashed arrow). Collectively, these pathways maintain genomic integrity throughout development and maintenance of the nervous system, and various neuropathology can result from their inactivation or attenuation.
In contrast to DNA DSBs, damage to only one DNA strand activates a distinct repair pathway termed single strand break repair (SSBR), and mutations in components of this pathway can also lead to human neurodegenerative syndromes. However, SSBR syndromes lack extra-neurological phenotypes, underscoring the unique importance of this pathway in the nervous system. Two prominent syndromes resulting from loss of DNA SSBR capacity are ataxia with oculomotor apraxia (AOA1) and spinocerebellar ataxia with axonal neuropathy (SCAN1). The syndromes result from respective defects in the DNA repair enzymes tyrosyl phosphodiesterase 1 (TDP1) which cleaves topoisomerase I-DNA complexes and other DNA 3’-termini, and Aprataxin (APTX) which removes 5’-adenylated DNA intermediates at the DNA break to allow ligation to proceed. We generated models for AOA1 and SCAN1 to further define the requirements for these factors during nervous system function [Nature443: 713-718, 2006; EMBO J. 26:4720-31, 2007]. In parallel, we also determined the role of a central SSBR factor, Xrcc1 and found that inactivation of Xrcc1 lead to the loss of a specific sub-type of cerebellar neuron (interneurons) that had not previously been linked to DNA damage, thus revealing a novel target in the cerebellum for DNA repair deficiency [NatureNeuroscience 2009, In Press].
Figure 4. Interneurons are missing in the Xrcc1Nes-cre cerebellum. (a) Comparative view of 7-week-old wild-type and Xrcc1Nes-cre cerebella after calbindin immunostaining; although smaller, histology of the mutant cerebellum was similar to that of the wild type. (b) Nissl staining at different ages indicates the presence of interneurons in the molecular layer (ML) of the control cerebellum (arrows) while interneurons are absent from the ML of Xrcc1Nes-cre mice.