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

X-irradiation of cells on glass slides has a dose doubling impact

Immunofluorescence detection of γH2AX foci is a widely used tool to quantify the induction and repair of DNA double-strand breaks (DSBs) induced by ionising radiation. We observed that X-irradiation of mammalian cells exposed on glass slides induced twofold higher foci numbers compared to irradiation with γ-rays. Here, we show that the excess γH2AX foci after X-irradiation are produced from secondary radiation particles generated from the irradiation of glass slides. Both 120 kV X-rays and 137Cs γ-rays induce ∼20 γH2AX foci per Gy in cells growing on thin (∼2 μm) plastic foils immersed in water. The same yield is obtained following γ-irradiation of cells growing on glass slides. However, 120 kV X-rays produce ∼40 γH2AX foci per Gy in cells growing on glass, twofold greater than obtained using cells irradiated on plastic surfaces. The same increase in γH2AX foci number is obtained if the plastic foil on which the cells are grown is irradiated on a glass slide. Thus, the physical proximity to the glass material and not morphological differences of cells growing on different surfaces accounts for the excess γH2AX foci. The increase in foci number depends on the energy and is considerably smaller for 25 kV relative to 120 kV X-rays, a finding which can be explained by known physical properties of radiation. The kinetics for the loss of foci, which is taken to represent the rate of DSB repair, as well as the Artemis dependent repair fraction, was similar following X- or γ-irradiation, demonstrating that DSBs induced by this range of treatments are repaired in an identical manner

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

ATM Release at Resected Double-Strand Breaks Provides Heterochromatin Reconstitution to Facilitate Homologous Recombination

Non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two main pathways for repairing DNA double-strand breaks (DSBs). During the G2 phase of the mammalian cell cycle, both processes can operate and chromatin structure is one important factor which determines DSB repair pathway choice. ATM facilitates the repair of heterochromatic DSBs by phosphorylating and inactivating the heterochromatin building factor KAP-1, leading to local chromatin relaxation. Here, we show that ATM accumulation and activity is strongly diminished at DSBs undergoing end-resection during HR. Such DSBs remain unrepaired in cells devoid of the HR factors BRCA2, XRCC3 or RAD51. Strikingly, depletion of KAP-1 or expression of phospho-mimic KAP-1 allows repair of resected DSBs in the absence of BRCA2, XRCC3 or RAD51 by an erroneous PARP-dependent alt-NHEJ process. We suggest that DSBs in heterochromatin elicit initial local heterochromatin relaxation which is reversed during HR due to the release of ATM from resection break ends. The restored heterochromatic structure facilitates HR and prevents usage of error-prone alternative processes

Understanding the limitations of radiation-induced cell cycle checkpoints

The DNA damage response pathways involve processes of double-strand break (DSB) repair and cell cycle checkpoint control to prevent or limit entry into S phase or mitosis in the presence of unrepaired damage. Checkpoints can function to permanently remove damaged cells from the actively proliferating population but can also halt the cell cycle temporarily to provide time for the repair of DSBs. Although efficient in their ability to limit genomic instability, checkpoints are not foolproof but carry inherent limitations. Recent work has demonstrated that the G1/S checkpoint is slowly activated and allows cells to enter S phase in the presence of unrepaired DSBs for about 4–6 h post irradiation. During this time, only a slowing but not abolition of S-phase entry is observed. The G2/M checkpoint, in contrast, is quickly activated but only responds to a level of 10–20 DSBs such that cells with a low number of DSBs do not initiate the checkpoint or terminate arrest before repair is complete. Here, we discuss the limitations of these checkpoints in the context of the current knowledge of the factors involved. We suggest that the time needed to fully activate G1/S arrest reflects the existence of a restriction point in G1-phase progression. This point has previously been defined as the point when mitogen starvation fails to prevent cells from entering S phase. However, cells that passed the restriction point can respond to DSBs, albeit with reduced efficiency

DNA repair synthesis and histone deposition partner during homologous recombination

Chromatin remodeling is critical for the regulation of the DNA damage response. We highlight findings from our recent study showing that the deposition of the histone variant H3.3 by the alpha-thalassemia mental retardation X-linked protein (ATRX) and the death domain associated protein (DAXX) chromatin remodeling complex regulates DNA repair synthesis during homologous recombination