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

DNA DSB repair pathway choice: an orchestrated handover mechanism

DNA double strand breaks (DSBs) are potential lethal lesions but can also lead to chromosome rearrangements, a step promoting carcinogenesis. DNA non-homologous end-joining (NHEJ) is the major DSB rejoining process and occurs in all cell cycle stages. Homologous recombination (HR) can additionally function to repair irradiation-induced two-ended DSBs in G2 phase. In mammalian cells, HR predominantly uses a sister chromatid as a template for DSB repair; thus HR functions only in late S/G2 phase. Here, we review current insight into the interplay between HR and NHEJ in G2 phase. We argue that NHEJ represents the first choice pathway, repairing approximately 80% of X-ray-induced DSBs with rapid kinetics. However, a subset of DSBs undergoes end resection and repair by HR. 53BP1 restricts resection, thereby promoting NHEJ. During the switch fromNHEJ to HR, 53BP1 is repositioned to the periphery of enlarged irradiation-induced foci (IRIF) via a BRCA1-dependent process. K63-linked ubiquitin chains, which also form at IRIF, are also repositioned as well as receptor-associated protein 80 (RAP80), a ubiquitin binding protein. RAP80 repositioning requires POH1, a proteasome component. Thus, the interfacing barriers to HR, 53BP1 and RAP80 are relieved by POH1 and BRCA1, respectively. Removal of RAP80 from the IRIF core is required for loss of the ubiquitin chains and 53BP1, and for efficient replication protein A foci formation. We propose that NHEJ is used preferentially to HR because it is a compact process that does not necessitate extensive chromatin changes in the DSB vicinity

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

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DNA double-strand breaks: their cellular and clinical impact?

DNA, the central store of our genetic information, constantly incurs damage from agents generated within the cell as well as chemicals or radiation that arise externally. Of the many different classes of damage, a DNA double-strand break (DSB) is arguably the most significant since, if unrepaired it can result in cell death and if misrepaired, it can cause chromosomal translocations, an early step in the aetiology of carcinogenesis. Endogenously generated reactive oxygen species primarily induce base damage and single strand breaks and it is unlikely that DNA DSBs are directly induced to any significant extent. However, DSBs may arise indirectly from two closely located single-strand breaks or during the repair of other lesions. They also arise when replication forks collapse, which may occur following the attempted replication of single-strand breaks or base damage. Indeed, a DSB is very likely the ultimate lesion induced by a wide range of DNA-damaging agents. The enhanced levels of endogenous chromosome breakage or chromosome rearrangements that have been observed in cells that fail to repair DSBs efficiently attests to the fact that they represent a relatively frequently encountered endogenous lesion (Karanjawala et al., 1999). Despite the constant onslaught of endogenous oxidative damage as well as frequently encountered exogenous DNA damage, genomic changes are a rare event and cells can undergo multiple rounds of replication without witnessing chromosomal alterations. This attests to the remarkable efficiency and evolutionary importance of the pathways that function in response to DSB induction

The C Terminus of Ku80 activates the DNA-dependent protein kinase catalytic subunit

Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining. Ku is thought to target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA-PKcs). We have carried out a 3′ deletion analysis of Ku80, the larger subunit of Ku, and shown that the C-terminal 178 amino acid residues are dispensable for DNA end-binding activity but are required for efficient interaction of Ku with DNA-PKcs. Cells expressing Ku80 proteins that lack the terminal 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J recombination. These cells have therefore acquired the phenotype of mouse SCID cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is required for DNA double-strand break rejoining and coding but not signal joint formation. To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line. This residue is conserved among species and lies outside of the previously reported Ku70-Ku80 interaction domain. The mutational change nonetheless abrogates the Ku70-Ku80 interaction and DNA end-binding activity

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

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