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

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

Patient radiation dose issues resulting from the use of CT in the UK

In this report, COMARE presents a comprehensive review of the radiation dose issues associated with CT scans in the UK. The implications of the increase in the numbers of CT scans in the UK are considered in the report, with focus on the number of younger patients undergoing CT scans, who have greater sensitivity to x-rays. The report provides an update on the radiation protection aspects of justification (balancing risk and benefit) and optimisation (balancing the risk from the radiation dose with the quality of the image)

Mutations in pericentrin cause Seckel syndrome with defective ATR-dependent DNA damage signaling

Large brain size is one of the defining characteristics of modern humans. Seckel syndrome (MIM 210600), a disorder of markedly reduced brain and body size, is associated with defective ATR-dependent DNA damage signaling. Only a single hypomorphic mutation of ATR has been identified in this genetically heterogeneous condition. We now report that mutations in the gene encoding pericentrin (PCNT)--resulting in the loss of pericentrin from the centrosome, where it has key functions anchoring both structural and regulatory proteins--also cause Seckel syndrome. Furthermore, we find that cells of individuals with Seckel syndrome due to mutations in PCNT (PCNT-Seckel) have defects in ATR-dependent checkpoint signaling, providing the first evidence linking a structural centrosomal protein with DNA damage signaling. These findings also suggest that other known microcephaly genes implicated in either DNA repair responses or centrosomal function may act in common developmental pathways determining human brain and body size

A coordinated DNA damage response promotes adult quiescent neural stem cell activation

Stem and differentiated cells frequently differ in their response to DNA damage, which can determine tissue sensitivity. By exploiting insight into the spatial arrangement of subdomains within the adult neural subventricular zone (SVZ) in vivo, we show distinct responses to ionising radiation (IR) between neural stem and progenitor cells. Further, we reveal different DNA damage responses between neonatal and adult neural stem cells (NSCs). Neural progenitors (transit amplifying cells and neuroblasts) but not NSCs (quiescent and activated) undergo apoptosis after 2 Gy IR. This response is cell type- rather than proliferationdependent and does not appear to be driven by distinctions in DNA damage induction or repair capacity. Moreover, exposure to 2 Gy IR promotes proliferation arrest and differentiation in the adult SVZ. These 3 responses are ataxia telangiectasia mutated (ATM)- dependent and promote quiescent NSC (qNSC) activation, which does not occur in the subdomains that lack progenitors. Neuroblasts arising post-IR derive from activated qNSCs rather than irradiated progenitors, minimising damage compounded by replication or mitosis. We propose that rather than conferring sensitive cell death, apoptosis is a form of rapid cell death that serves to remove damaged progenitors and promote qNSC activation. Significantly, analysis of the neonatal (P5) SVZ reveals that although progenitors remain sensitive to apoptosis, they fail to efficiently arrest proliferation. Consequently, their repopulation occurs rapidly from irradiated progenitors rather than via qNSC activation

Identification of the first ATRIP-deficient patient and novel mutations in ATR define a clinical spectrum for ATR-ATRIP Seckel Syndrome

A homozygous mutational change in the Ataxia-Telangiectasia and RAD3 related (ATR) gene was previously reported in two related families displaying Seckel Syndrome (SS). Here, we provide the first identification of a Seckel Syndrome patient with mutations in ATRIP, the gene encoding ATR-Interacting Protein (ATRIP), the partner protein of ATR required for ATR stability and recruitment to the site of DNA damage. The patient has compound heterozygous mutations in ATRIP resulting in reduced ATRIP and ATR expression. A nonsense mutational change in one ATRIP allele results in a C-terminal truncated protein, which impairs ATR-ATRIP interaction; the other allele is abnormally spliced. We additionally describe two further unrelated patients native to the UK with the same novel, heterozygous mutations in ATR, which cause dramatically reduced ATR expression. All patient-derived cells showed defective DNA damage responses that can be attributed to impaired ATR-ATRIP function. Seckel Syndrome is characterised by microcephaly and growth delay, features also displayed by several related disorders including Majewski (microcephalic) osteodysplastic primordial dwarfism (MOPD) type II and Meier-Gorlin Syndrome (MGS). The identification of an ATRIP-deficient patient provides a novel genetic defect for Seckel Syndrome. Coupled with the identification of further ATR-deficient patients, our findings allow a spectrum of clinical features that can be ascribed to the ATR-ATRIP deficient sub-class of Seckel Syndrome. ATR-ATRIP patients are characterised by extremely severe microcephaly and growth delay, microtia (small ears), micrognathia (small and receding chin), and dental crowding. While aberrant bone development was mild in the original ATR-SS patient, some of the patients described here display skeletal abnormalities including, in one patient, small patellae, a feature characteristically observed in Meier-Gorlin Syndrome. Collectively, our analysis exposes an overlapping clinical manifestation between the disorders but allows an expanded spectrum of clinical features for ATR-ATRIP Seckel Syndrome to be define

Protective role of vitamin B6 (PLP) against DNA damage in Drosophila models of type 2 diabetes

Growing evidence shows that improper intake of vitamin B6 increases cancer risk and several studies indicate that diabetic patients have a higher risk of developing tumors. We previously demonstrated that in Drosophila the deficiency of Pyridoxal 5' phosphate (PLP), the active form of vitamin B6, causes chromosome aberrations (CABs), one of cancer prerequisites, and increases hemolymph glucose content. Starting from these data we asked if it was possible to provide a link between the aforementioned studies. Thus, we tested the effect of low PLP levels on DNA integrity in diabetic cells. To this aim we generated two Drosophila models of type 2 diabetes, the first by impairing insulin signaling and the second by rearing flies in high sugar diet. We showed that glucose treatment induced CABs in diabetic individuals but not in controls. More interestingly, PLP deficiency caused high frequencies of CABs in both diabetic models demonstrating that hyperglycemia, combined to reduced PLP level, impairs DNA integrity. PLP-depleted diabetic cells accumulated Advanced Glycation End products (AGEs) that largely contribute to CABs as α-lipoic acid, an AGE inhibitor, rescued not only AGEs but also CABs. These data, extrapolated to humans, indicate that low PLP levels, impacting on DNA integrity, may be considered one of the possible links between diabetes and cancer

Cellular Radiosensitivity: How much better do we understand it?

Purpose: Ionizing radiation exposure gives rise to a variety of lesions in DNA that result in genetic instability and potentially tumorigenesis or cell death. Radiation extends its effects on DNA by direct interaction or by radiolysis of H2O that generates free radicals or aqueous electrons capable of interacting with and causing indirect damage to DNA. While the various lesions arising in DNA after radiation exposure can contribute to the mutagenising effects of this agent, the potentially most damaging lesion is the DNA double strand break (DSB) that contributes to genome instability and/or cell death. Thus in many cases failure to recognise and/or repair this lesion determines the radiosensitivity status of the cell. DNA repair mechanisms including homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to protect cells against DNA DSB. Mutations in proteins that constitute these repair pathways are characterised by radiosensitivity and genome instability. Defects in a number of these proteins also give rise to genetic disorders that feature not only genetic instability but also immunodeficiency, cancer predisposition, neurodegeneration and other pathologies. Conclusions: In the past fifty years our understanding of the cellular response to radiation damage has advanced enormously with insight being gained from a wide range of approaches extending from more basic early studies to the sophisticated approaches used today. In this review we discuss our current understanding of the impact of radiation on the cell and the organism gained from the array of past and present studies and attempt to provide an explanation for what it is that determines the response to radiation