Radiation induced DNA damage responses

The amazing feature of ionising radiation (IR) as a DNA damaging agent is the range of lesions it induces. Such lesions include base damage, single strand breaks (SSBs), double strand breaks (DSBs) of varying complexity and DNA cross links. A range of DNA damage response mechanisms operate to help maintain genomic stability in the face of such damage. Such mechanisms include pathways of DNA repair and signal transduction mechanisms. Increasing evidence suggests that these pathways operate co-operatively. In addition, the relative impact of one mechanism over another most probably depends upon the cell cycle phase and tissue type. Here, the distinct damage response pathways are reviewed and the current understanding of the interplay between them is considered. Since DNA DSBs are the major lethal lesion induced by IR, the focus lies in the mechanisms responding to direct or indirectly induced DSBs

[Editorial] DNA non-homologous end-joining enters the resection arena

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The pendulum of the Ku-Ku clock

Canonical DNA non-homologous end-joining (c-NHEJ) and homologous recombination (HR), the two major DNA double-strand break (DSB) repair pathways, have long been depicted as competitors, fighting a race to rejoin DSBs. In human cells, Ku, an upstream component of NHEJ, is highly abundant and has exquisite end-binding capacity. Emerging evidence has suggested that Ku is the first protein binding most, if not all, DSBs, and creates a block to resection. Although most c-NHEJ proceeds without resection, recent studies have provided strong evidence for a process of resection-dependent c-NHEJ, that repairs a subset of DSBs. HR also repairs a subset of two-ended DSBs in G2 phase and processes one-ended DSBs that arise following replication fork stalling or collapse to promote replication restart. HR also necessitates end-resection. This raises the question of how end-resection takes place despite Ku’s avid end-binding capacity. Insight into this enigma has been gained from the analysis of DSBs generated by Spo11 or TOP2, which create protein-bridged DSBs. The progression of repair by HR or NHEJ requires removal of the end-blocking lesions. The MRE11-RAD50-NBS1 (MRN) complex, CtIP and EXO1 play critical roles in this process. Here, we review our current understanding of how resection arises at lesions blocked by covalently bound Spo11 or TOP2 or following Ku binding, which effectively creates a distinct resection-blocking lesion due to its avid end-binding activity and abundance. Our review reveals that Ku plays an active role in determining pathway choice and exposes similarities yet distinctions in the progression of resection that is suited to the optimal repair pathway choice

In vivo sensitivity of the embryonic and adult neural stem cell compartments to low-dose radiation

The embryonic brain is radiation-sensitive, with cognitive deficits being observed after exposure to low radiation doses. Exposure of neonates to radiation can cause intracranial carcinogenesis. To gain insight into the basis underlying these outcomes, we examined the response of the embryonic, neonatal and adult brain to low-dose radiation, focusing on the neural stem cell compartments. This review summarizes our recent findings. At E13.5-14.5 the embryonic neocortex encompasses rapidly proliferating stem and progenitor cells. Exploiting mice with a hypomorphic mutation in DNA ligase IV (Lig4(Y288C) ), we found a high level of DNA double-strand breaks (DSBs) at E14.5, which we attribute to the rapid proliferation. We observed endogenous apoptosis in Lig4(Y288C) embryos and in WT embryos following exposure to low radiation doses. An examination of DSB levels and apoptosis in adult neural stem cell compartments, the subventricular zone (SVZ) and the subgranular zone (SGZ) revealed low DSB levels in Lig4(Y288C) mice, comparable with the levels in differentiated neuronal tissues. We conclude that the adult SVZ does not incur high levels of DNA breakage, but sensitively activates apoptosis; apoptosis was less sensitively activated in the SGZ, and differentiated neuronal tissues did not activate apoptosis. P5/P15 mice showed intermediate DSB levels, suggesting that DSBs generated in the embryo can be transmitted to neonates and undergo slow repair. Interestingly, this analysis revealed a stage of high endogenous apoptosis in the neonatal SVZ. Collectively, these studies reveal that the adult neural stem cell compartment, like the embryonic counterpart, can sensitively activate apoptosis

Chromosome breakage after G2 checkpoint release

DNA double-strand break (DSB) repair and checkpoint control represent distinct mechanisms to reduce chromosomal instability. Ataxia telangiectasia (A-T) cells have checkpoint arrest and DSB repair defects. We examine the efficiency and interplay of ATM's G2 checkpoint and repair functions. Artemis cells manifest a repair defect identical and epistatic to A-T but show proficient checkpoint responses. Only a few G2 cells enter mitosis within 4 h after irradiation with 1 Gy but manifest multiple chromosome breaks. Most checkpoint-proficient cells arrest at the G2/M checkpoint, with the length of arrest being dependent on the repair capacity. Strikingly, cells released from checkpoint arrest display one to two chromosome breaks. This represents a major contribution to chromosome breakage. The presence of chromosome breaks in cells released from checkpoint arrest suggests that release occurs before the completion of DSB repair. Strikingly, we show that checkpoint release occurs at a point when approximately three to four premature chromosome condensation breaks and approximately 20 gammaH2AX foci remain

An imperfect G2M checkpoint contributes to chromosome instability following irradiation of S and G2 phase cells

DNA double strand break (DSB) repair and checkpoint control represent two major mechanisms that function to reduce chromosomal instability following ionising irradiation (IR). Ataxia telangiectasia (A-T) cells have long been known to have defective checkpoint responses. Recent studies have shown that they also have a DSB repair defect following IR raising the issue of how ATM’s repair and checkpoint functions interplay to maintain chromosomal stability. A-T and Artemis cells manifest an identical and epistatic repair defect throughout the cell cycle demonstrating that ATM’s major repair defect following IR represents Artemis-dependent end-processing. Artemis cells show efficient G2/M checkpoint induction and a prolonged arrest relative to normal cells. Following irradiation of G2 cells, this checkpoint is dependent on ATM and A-T cells fail to show checkpoint arrest. In contrast, cells irradiated during S phase initiate a G2/M checkpoint which is independent of ATM and, significantly, both Artemis and A-T cells show a prolonged arrest at the G2/M checkpoint likely reflecting their repair defect. Strikingly, the G2/M checkpoint is released before the completion of repair when approximately 10-20 DSBs remain both for S phase and G2 phase irradiated cells. This defined sensitivity level of the G2/M checkpoint explains the prolonged arrest in repair-deficient relative to normal cells and provides a conceptual framework for the co-operative phenotype between checkpoint and repair functions in maintaining chromosomal stability

ATR promotes cilia signalling: links to developmental impacts

Mutations in ATR (ataxia telangiectasia and RAD3-related) cause Seckel syndrome (ATR-SS), a microcephalic primordial dwarfism disorder. Hitherto, the clinical manifestation of ATR deficiency has been attributed to its canonical role in DNA damage response signalling following replication fork stalling/collapse. Here, we show that ATR regulates cilia-dependent signalling in a manner that can be uncoupled from its function during replication. ATR-depleted or patient-derived ATR-SS cells form cilia of slightly reduced length but are dramatically impaired in cilia-dependent signalling functions, including growth factor and Sonic hedgehog signalling. To better understand the developmental impact of ATR loss of function, we also used zebrafish as a model. Zebrafish embryos depleted of Atr resembled ATR-SS morphology, showed a modest but statistically significant reduction in cilia length and other morphological features indicative of cilia dysfunction. Additionally, they displayed defects in left-right asymmetry including ambiguous expression of southpaw, incorrectly looped hearts and randomized localization of internal organs including the pancreas, features typically conferred by cilia dysfunction. Our findings reveal a novel role for ATR in cilia signalling distinct from its canonical function during replication and strengthen emerging links between cilia function and development