Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G2/M Checkpoint Arrest

ATM-dependent initiation of the radiation-induced G₂/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G₂ phase are repaired by DNA nonhomologous end joining (NHEJ), while ∼15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G₂/M checkpoint is maintained in irradiated G₂ cells, in light of our current understanding of G₂ phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1^−/− and MDC1^−/− cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.DNA double-strand breaks (DSBs) activate the DNA damage response (DDR), a coordinated process that functions to enhance survival and maintain genomic stability. The DDR includes pathways of DSB repair and a signal transduction response that activates apoptosis and cell cycle checkpoint arrest and influences DSB repair (15). DNA nonhomologous end joining (NHEJ) and homologous recombination (HR) represent the major DSB repair mechanisms, NHEJ being the major mechanism in G₀/G₁, while both processes function in G₂ (9, 32). Ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) are related phosphoinositol 3-kinase-like kinases (PIKKs) that regulate the DNA damage signaling response. ATM is activated by DSBs, while ATR is activated at single-strand (ss) regions of DNA via a process that involves ATRIP-replication protein A (RPA)-ssDNA association. Ionizing radiation (IR) induces DSBs, base damage, and ss nicks. Since neither base damage nor ss nicks activate ATR, IR-induced signaling in the G₁ and G₂ phases is predominantly ATM dependent (3, 29). In S phase, ATR can be activated by both endogenous and exogenously induced lesions following replication fork stalling/collapse (8).Recent work has shown that in G₂ phase, DSBs can undergo resection via an ATM-dependent process generating ssDNA regions that can activate ATR following RPA association (11). ATR activation at resected DSBs is coupled to loss of ATM activation (11). Although ATM and ATR share overlapping substrates, there is specificity in their signaling to the transducer kinases; ATM uniquely phosphorylates Chk2, while ATR phosphorylates Chk1. Phosphorylation of either Chk1 or Chk2 causes their activation. Critical targets of Chk1/Chk2 are the Cdc25 phosphatases, which regulate the cyclin-dependent kinases (Cdks), including Cdk1, the regulator of mitotic entry (18). Collectively, these studies suggest that two components of ATM-dependent signaling to the G₂/M checkpoint machinery can occur: ATM-Chk2 signaling at unresected DSBs and ATM-ATR-Chk1 signaling at resected DSBs.Although much is known about the mechanism leading to G₂/M checkpoint activation, few studies have addressed how arrest is maintained and how release coordinates with the status of DSB repair. We examine here the maintenance of checkpoint arrest during the immediate phase of DSB repair. We do not address the issue of checkpoint adaptation, a distinct phenomenon which occurs after prolonged checkpoint arrest (22). Further, we focus on the process maintaining arrest in irradiated G₂-phase cells and do not consider how arrest is maintained in irradiated S-phase cells that progress into G₂ phase. (Previous studies have shown that while G₂/M arrest is ATM dependent at early times post-IR, at later times it becomes ATR dependent as S-phase cells progress into G₂ phase [2, 33].) To focus on mechanisms maintaining ATM-dependent signaling in G₂-phase cells, we use aphidicolin (APH) to prevent S-phase cells from progressing into G₂ during analysis. We, thus, examine checkpoint maintenance in cells irradiated in G₂ phase and do not evaluate arrest regulated by ATR following replication fork stalling. The basis for our work stems from two recent advances. First, we evaluate the impact of ATM-mediated ATR activation in the light of recent findings that resection occurs in G₂ phase (11). Second, we consider the finding that NHEJ represents the major DSB repair mechanism in G₂ and that a 15 to 20% subset of DSBs, representing those that are rejoined with slow kinetics in an ATM-dependent manner, undergo resection and repair by HR (3, 25). Thus, contrary to the notion that HR represents the major DSB repair pathway in G₂ phase, it repairs only 15 to 20% of X- or gamma-ray-induced DSBs and represents the slow component of DSB repair in G₂ phase. Given these findings, several potential models for how checkpoint arrest is maintained in G₂ can be envisaged. A simple model is that the initial signal generated by IR is maintained for a defined time to allow for DSB repair. Such a model appears to explain the kinetics of checkpoint signaling in fission yeast after moderate IR (17). In mammalian cells, the duration of arrest depends on dose and DSB repair capacity (6). Thus, it is possible that the status of ongoing repair is communicated to the checkpoint machinery to coordinate timely release with the process of DSB repair. Here, we consider the impact of resection leading to ATM-ATR-Chk1 signaling versus ATM-Chk2 signaling from nonresected DSBs and how they interplay to maintain rather than initiate checkpoint arrest.Mediator proteins, including 53BP1 and MDC1, assemble at DSBs in an ATM-dependent manner, but their roles in the DDR are unclear. Cells lacking 53BP1 or MDC1 are proficient in checkpoint initiation after moderate IR doses, leading to the suggestion that these proteins are required for amplification of the ATM signal after exposure to low doses but are dispensable after high doses, when a robust signal is generated, even in their absence (7, 16, 28, 31). Despite their apparent subtle role in ATM signaling, cells lacking these mediator proteins display significant genomic instability (19). We thus also examine whether the mediator proteins contribute to the maintenance of checkpoint arrest.We identify two ATM-dependent processes that contribute to the maintenance of checkpoint arrest in G₂-phase cells: (i) ATR-Chk1 activation at resected DSBs and (ii) a process that involves sustained signaling from ATM to Chk2 at unrepaired DSBs. Further, we show that 53BP1 and MDC1 are required for maintaining checkpoint arrest, even following exposure to high radiation doses due to roles in ATR-Chk1 activation and sustained ATM-Chk2 signaling, and that this contributes to their elevated genomic instability.     

Nutrient dynamics at the interface between surface waters and groundwaters

1. The surface water/groundwater (SW/GW) interface is a crucial control point for lateral nutrient fluxes between uplands and aquatic ecosystems and for upstream/downstream (longitudinal) processes in lotic ecosystems. 2. Hydrological and biogeochemical dynamics of the SW/GW ecotone are linked to the degree of channel constraint and the sediment characteristics of the floodplain and stream bed. 3. The availability of specific chemical forms of electron donors and electron acceptors affects the spatial distribution of biogeochemical processes at the SW/GW interface. Temporal change in discharge is also a major factor affecting the rate and extent of these processes. 4. The magnitude of SW/GW interactions in lotic ecosystems is predicted to be a major determinant of solute retention. Channel morphology, stream bed composition and discharge are predicted to be important controls on SW/GW interactions. 5. Interdisciplinary research involving hydrologists, geomorphologists, aquatic ecologists, microbial ecologists and landscape ecologists is needed to further our present understanding of this critical interface linking terrestrial and aquatic ecosystems.     

Factors affecting ammonium uptake in streams – an inter-biome perspective

1. The Lotic Intersite Nitrogen eXperiment (LINX) was a coordinated study of the relationships between North American biomes and factors governing ammonium uptake in streams. Our objective was to relate inter‐biome variability of ammonium uptake to physical, chemical and biological processes. 2. Data were collected from 11 streams ranging from arctic to tropical and from desert to rainforest. Measurements at each site included physical, hydraulic and chemical characteristics, biological parameters, whole‐stream metabolism and ammonium uptake. Ammonium uptake was measured by injection of ¹⁵N‐ammonium and downstream measurements of ¹⁵N‐ammonium concentration. 3. We found no general, statistically significant relationships that explained the variability in ammonium uptake among sites. However, this approach does not account for the multiple mechanisms of ammonium uptake in streams. When we estimated biological demand for inorganic nitrogen based on our measurements of in‐stream metabolism, we found good correspondence between calculated nitrogen demand and measured assimilative nitrogen uptake. 4. Nitrogen uptake varied little among sites, reflecting metabolic compensation in streams in a variety of distinctly different biomes (autotrophic production is high where allochthonous inputs are relatively low and vice versa). 5. Both autotrophic and heterotrophic metabolism require nitrogen and these biotic processes dominate inorganic nitrogen retention in streams. Factors that affect the relative balance of autotrophic and heterotrophic metabolism indirectly control inorganic nitrogen uptake.     

Stimulation of vitellogenin production by methoprene in prepupae and pupae of manduca sexta

Denaturing electrophoresis of hemolymph from prepupae of M. sexta showed trace amounts of polypeptides with mobilities corresponding to those of vitellogenin (Vg) apoproteins from adult females. Absence of the polypeptides in allatectomized insects suggested regulation by juvenile hormone (JH). Daily administration of 10 μg of the JH analog methoprene from day 4 of the fifth stage to day 0 of the pupal stage caused accumulation of these polypeptides. They were identified as apovitellogenins (apoVgs) immunochemically with Vg antiserum. Stimulation of Vg in response to methoprene varied with age. In all cases, day 0 female pupae were highly responsive. Vg synthesis was not stimulated when pupae were injected with 20-hydroxyecdysone (20-HE) in addition to methoprene. Methoprene-stimulated Vg synthesis was also abolished by inhibitors of mRNA or protein synthesis (α-amanitin, actinomycin, cycloheximide). This result indicated that methoprene-stimulated Vg accumulation requires gene expression. A Vg cDNA (2.1 kb) obtained by immunoscreening of the λgt 11 library, when used as a radiolabelled probe, hybridized with a 5.1 kb mRNA from total RNA of female fat body. It also hybridized with fat body RNA of normal prepupae and methoprene treated day 0 pupae but not with that of early fifth instars or solvent control pupae. The results indicate that the trace amounts of Vg found in prepupal stages are due to a weak expression of the Vg gene, which is stimulated by JH and repressed by 20-HE. © 1994 Wiley-Liss, Inc.     

Simultaneous measures of contraction and intracellular calcium in single, cultured smooth muscle cells

Summary Simple methods are presented for quantitating contraction and intracellular calcium simultaneously in single, cultured smooth muscle cells. These methods are the first to demonstrate that reliable velocities of cell shortening can be measured in cultured smooth muscle cells and that cells in vitro exhibit shortening velocities comparable to those measured in the fastest phasic muscles in situ. Temporal relationships between changes in intracellular calcium and shortening within single cells were determined with a resolution of 100 ms and were consistent with measures in more “classical” preparations. Intracellular calcium rose quickly and transiently 10-fold above the basal level of 80–90 nM in response to the muscarinic agonist, carbachol. Shortening of the cells occurred 200 ms after intracellular calcium began to rise. The sensitivity and reliability of these methods allowed the effects of different stimuli to be easily resolved. The present report demonstrates that genuine contractility need not be ignored in cultured smooth muscle cells and that the temporal relations between shortening and intracellular calcium mobilization can be quantitatively assessed in controlled in vitro environments.     

Minimum sample sizes for identifying chromosomal fragile sites from individuals: Monte Carlo estimation

A Monte Carlo simulation procedure was used to estimate the exact level of the standardized X ² test statistic (X _s ²) for randomness in the FSM methodology for the identification of fragile sites from chromosomal breakage data for single individuals. A random-number generator was used to simulate 10 000 chromosomal breakage data sets, each corresponding to the null hypothesis of no fragile sites for numbers of chromosomal breaks (n) from 1 to 2000 and at three levels of chromosomal band resolution (k). The reliability of the test was assessed by comparisons of the empirical and nominal α levels for each of the corresponding values of n and k. These analyses indicate that the sparse and discrete nature of chromosomal breakage data results in large and unpredictable discrepancies between the empirical and nominal α levels when fragile site identifications are based on small numbers of breaks (n < 0.5 k). With n≥ 0.5 k, the distribution of X _s ² appears to be stable and non-significant differences in the empirical and nominal α levels are generally obtained. These results are inherent to the nature of the data and are, therefore, relevant to any statistical model for the identification of fragile sites from chromosomal breakage data. For FSM identification of fragile sites at α = 0.05, we suggest that n≥ 0.5 k is the minimum reliable number of mapped chromosomal breaks per individual. Received: 28 April 1997 / Accepted: 1 July 1997