Tom Stiff

ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation.

H2AX phosphorylation is an early step in the response to DNA damage. It is widely accepted that ATM (ataxia telangiectasia mutated protein) phosphorylates H2AX in response to DNA double-strand breaks (DSBs). Whether DNA-dependent protein kinase (DNA-PK) plays any role in this response is unclear. Here, we show that H2AX phosphorylation after exposure to ionizing radiation (IR) occurs to similar extents in human fibroblasts and in mouse embryo fibroblasts lacking either DNA-PK or ATM but is ablated in ATM-deficient cells treated with LY294002, a drug that specifically inhibits DNA-PK. Additionally, we show that inactivation of both DNA-PK and ATM is required to ablate IR-induced H2AX phosphorylation in chicken cells. We confirm that H2AX phosphorylation induced by DSBs in nonreplicating cells is ATR (ataxia telangiectasia and Rad3-related protein) independent. Taken together, we conclude that under most normal growth conditions, IR-induced H2AX phosphorylation can be carried out by ATM and DNA-PK in a redundant, overlapping manner. In contrast, DNA-PK cannot phosphorylate other proteins involved in the checkpoint response, including chromatin-associated Rad17. However, by phosphorylating H2AX, DNA-PK can contribute to the presence of the damage response proteins MDC1 and 53BP1 at the site of the DSB.

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.

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 ATMs 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 ∼20 γH2AX foci remain.

Mutations in ORC1, encoding the largest subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome.

Studies into disorders of extreme growth failure (for example, Seckel syndrome and Majewski osteodysplastic primordial dwarfism type II) have implicated fundamental cellular processes of DNA damage response signaling and centrosome function in the regulation of human growth. Here we report that mutations in ORC1, encoding a subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome. We establish that these mutations disrupt known ORC1 functions including pre-replicative complex formation and origin activation. ORC1 deficiency perturbs S-phase entry and S-phase progression. Additionally, we show that Orc1 depletion in zebrafish is sufficient to markedly reduce body size during rapid embryonic growth. Our data suggest a model in which ORC1 mutations impair replication licensing, slowing cell cycle progression and consequently impeding growth during development, particularly at times of rapid proliferation. These findings establish a novel mechanism for the pathogenesis of microcephalic dwarfism and show a surprising but important developmental impact of impaired origin licensing.

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 defined.

Deficiency in origin licensing proteins impairs cilia formation: implications for the aetiology of meier-gorlin syndrome

Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA–mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency.

Replication independent ATR signalling leads to G2/M arrest requiring Nbs1, 53BP1 and MDC1

Ataxia telangiectasia and Rad3-related (ATR) is a phosphoinositol-3-kinase like kinase (PIKK) that initiates a signal transduction response to replication fork stalling. Defects in ATR signalling have been reported in several disorders characterized by microcephaly and growth delay. Here, we gain insight into factors influencing the ATR signalling pathway and consider how they can be exploited for diagnostic purposes. Activation of ATR at stalled replication forks leads to intra-S and G2/M phase checkpoint arrest. ATR also phosphorylates gamma-H2AX at single-stranded (ss) DNA regions generated during nucleotide excision repair (NER) in non-replicating cells, but the critical analysis of any functional consequence has not been reported. Here, we show that UV irradiation of G2 phase cells causes ATR-dependent but replication-independent G2/M checkpoint arrest. This process requires the Nbs1 N-terminus encompassing the FHA and BRCT domains but not the Nbs1 C-terminus in contrast to ATM-dependent activation of G2/M arrest in response to ionizing radiation. Thus, Nbs1 has a function in ATR signalling in a manner distinct to any role at stalled replication forks. Replication-independent ATR signalling also requires the mediator proteins, 53BP1 and MDC1, providing direct evidence for their role in ATR signalling, but not H2AX. Finally, the process is activated in Cockaynes syndrome but not Xeroderma pigmentosum group A cells providing evidence that ssDNA regions generated during NER are the ATR-pathway-specific activating lesion. Replication-independent G2/M checkpoint arrest represents a suitable assay to specifically identify patients with defective ATR signalling, including Seckel syndrome, Nijmegen breakage syndrome and MCPH-1-dependent primary microcephaly.

DNA double-strand break repair within heterochromatic regions.

DNA DSBs (double-strand breaks) represent a critical lesion for a cell, with misrepair being potentially as harmful as lack of repair. In mammalian cells, DSBs are predominantly repaired by non-homologous end-joining or homologous recombination. The kinetics of repair of DSBs can differ widely, and recent studies have shown that the higher-order chromatin structure can dramatically affect the pathway utilized, the rate of repair and the genetic factors required for repair. Studies of the repair of DSBs arising within heterochromatic DNA regions have provided insight into the constraints that higher-order chromatin structure poses on repair and the processing that is uniquely required for the repair of such DSBs. In the present paper, we provide an overview of our current understanding of the process of heterochromatic DSB repair in mammalian cells and consider the evolutionary conservation of the processes.

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.

Checkpoint Control Following Radiation Exposure

The DNA damage response involves processes of DNA repair and signal transduction pathways. DNA double-strand breaks (DSBs) are the most biologically significant lesion induced by ionizing radiation (IR). DNA nonhomologous end-joining and homologous recombination represent the major DSB repair pathways and ataxia telangiectasia mutated (ATM) lies at the heart of the DSB signaling response. However, ATM and Rad3 related (ATR) can also be activated in S and G2 phase following IR exposure. A major end point of damage response signaling is the activation of cell cycle checkpoint arrest. In addition to the initiation of checkpoint arrest, recent studies have demonstrated that the signaling pathway monitors the progress of DSB repair to ensure timely checkpoint release, a process called the maintenance of checkpoint arrest. In this chapter, we overview DNA damage-induced cell cycle checkpoint arrest following exposure to IR. We discuss how cell cycle stage impacts upon the roles of ATM and ATR, how they influence the initiation and maintenance of checkpoint signaling, and the interface between checkpoint arrest and DSB repair. We evaluate current insight into the sensitivity of the processes and the impact of higher order chromatin structure on damage response signaling.