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At least 1,027 records · Page 57Linked to original sources
PubMed2005

Mutation in the 5' alternatively spliced region of the XNP/ATR-X gene causes Chudley-Lowry syndrome.

The Chudley-Lowry syndrome (ChLS, MIM 309490) is an X-linked recessive condition characterized by moderate to severe mental retardation, short stature, mild obesity, hypogonadism, and distinctive facial features characterized by depressed nasal bridge, anteverted nares, inverted-V-shaped upper lip, and macrostomia. The original Chudley-Lowry family consists of three affected males in two generations. Linkage analysis had localized the gene to a large interval, Xp21-Xq26 and an obligate carrier was demonstrated to have highly skewed X inactivation. The combination of the clinical phenotype, consistent with that of the patients with ATR-X syndrome, the skewed X-inactivation pattern in a carrier female, as well as the mapping interval including band Xq13.3, prompted us to consider the XNP/ATR-X gene being involved in this syndrome. Using RT-PCR analysis, we screened the entire XNP/ATR-X gene and found a mutation in exon 2 (c.109C > T) giving rise to a stop codon at position 37 (p.R37X). Western blot and immunocytochemical analyses using a specific monoclonal antibody directed against XNP/ATR-X showed the protein to be present in lymphoblastoid cells from one affected male, despite the premature stop codon. To explain these discordant results, we further analyzed the 5' region of the XNP/ATR-X gene and found three alternative transcripts, which differ in the presence or absence of exon 2, and the length of exon 1. Our data suggest that ChLS is allelic to the ATR-X syndrome with its less severe phenotype being due to the presence of some XNP/ATR-X protein.

Alternative Splicing↗
PubMed2004

Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling.

Human Rad17 (hRad17) is centrally involved in the activation of cell-cycle checkpoints by genotoxic agents or replication stress. Here we identify hMCM7, a core component of the DNA replication apparatus, as a novel hRad17-interacting protein. In HeLa cells, depletion of either hRad17 or hMCM7 with small-interfering RNA suppressed ultraviolet (UV) light- or aphidicolin-induced hChk1 phosphorylation, and abolished UV-induced S-phase checkpoint activation. Similar results were obtained after transfection of these cells with a fusion protein containing the hMCM7-binding region of hRad17. The hMCM7-depleted cells were also defective for the formation of ATR-containing nuclear foci after UV irradiation, suggesting that hMCM7 is required for stable recruitment of ATR to damaged DNA. These results demonstrate that hMCM7 plays a direct role in the transmission of DNA damage signals from active replication forks to the S-phase checkpoint machinery in human cells.

Aphidicolin↗
PubMed2000

Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator.

Bcl-2 family protein including anti-apoptotic (Bcl-2) or pro-apoptotic (Bax) members can form ion channels when incorporated into synthetic lipid bilayers. This contrasts with the observation that Bcl-2 stabilizes the mitochondrial membrane barrier function and inhibits the permeability transition pore complex (PTPC). Here we provide experimental data which may explain this apparent paradox. Bax and adenine nucleotide translocator (ANT), the most abundant inner mitochondrial membrane protein, can interact in artificial lipid bilayers to yield an efficient composite channel whose electrophysiological properties differ quantitatively and qualitatively from the channels formed by Bax or ANT alone. The formation of this composite channel can be observed in conditions in which Bax protein alone has no detectable channel activity. Cooperative channel formation by Bax and ANT is stimulated by the ANT ligand atractyloside (Atr) but inhibited by ATP, indicating that it depends on the conformation of ANT. In contrast to the combination of Bax and ANT, ANT does not form active channels when incorporated into membranes with Bcl-2. Rather, ANT and Bcl-2 exhibit mutual inhibition of channel formation. Bcl-2 prevents channel formation by Atr-treated ANT and neutralizes the cooperation between Bax and ANT. Our data are compatible with a ménage à trois model of mitochondrial apoptosis regulation in which ANT, the likely pore forming protein within the PTPC, interacts with Bax or Bcl-2 which influence its pore forming potential in opposing manners.

Animals↗
PubMed2001

Regulation of the G2/M transition by p53.

p53 protects mammals from neoplasia by inducing apoptosis, DNA repair and cell cycle arrest in response to a variety of stresses. p53-dependent arrest of cells in the G1 phase of the cell cycle is an important component of the cellular response to stress. Here we review recent evidence that implicates p53 in controlling entry into mitosis when cells enter G2 with damaged DNA or when they are arrested in S phase due to depletion of the substrates required for DNA synthesis. Part of the mechanism by which p53 blocks cells at the G2 checkpoint involves inhibition of Cdc2, the cyclin-dependent kinase required to enter mitosis. Cdc2 is inhibited simultaneously by three transcriptional targets of p53, Gadd45, p21, and 14-3-3 sigma. Binding of Cdc2 to Cyclin B1 is required for its activity, and repression of the cyclin B1 gene by p53 also contributes to blocking entry into mitosis. p53 also represses the cdc2 gene, to help ensure that cells do not escape the initial block. Genotoxic stress also activates p53-independent pathways that inhibit Cdc2 activity, activation of the protein kinases Chk1 and Chk2 by the protein kinases Atm and Atr. Chk1 and Chk2 inhibit Cdc2 by inactivating Cdc25, the phosphatase that normally activates Cdc2. Chk1, Chk2, Atm and Atr also contribute to the activation of p53 in response to genotoxic stress and therefore play multiple roles. p53 induces transcription of the reprimo, B99, and mcg10 genes, all of which contribute to the arrest of cells in G2, but the mechanisms of cell cycle arrest by these genes is not known. Repression of the topoisomerase II gene by p53 helps to block entry into mitosis and strengthens the G2 arrest. In summary, multiple overlapping p53-dependent and p53-independent pathways regulate the G2/M transition in response to genotoxic stress.

14-3-3 Proteins↗
PubMed2003

Werner's syndrome protein is phosphorylated in an ATR/ATM-dependent manner following replication arrest and DNA damage induced during the S phase of the cell cycle.

Werner's syndrome (WS) is an autosomal recessive disorder, characterized at the cellular level by genomic instability in the form of variegated translocation mosaicism and extensive deletions. Individuals with WS prematurely develop multiple age-related pathologies and exhibit increased incidence of cancer. WRN, the gene defective in WS, encodes a 160-kDa protein (WRN), which has 3'-5'exonuclease, DNA helicase and DNA-dependent ATPase activities. WRN-defective cells are hypersensitive to certain genotoxic agents that cause replication arrest and/or double-strand breaks at the replication fork, suggesting a pivotal role for WRN in the protection of the integrity of the genoma during the DNA replication process. Here, we show that WRN is phosphorylated through an ATR/ATM dependent pathway in response to replication blockage. However, we provide evidence that WRN phosphorylation is not essential for its subnuclear relocalization after replication arrest. Finally, we show that WRN and ATR colocalize after replication fork arrest, suggesting that WRN and the ATR kinase collaborate to prevent genome instability during the S phase.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2001

FTIR sensing of inhomogeneous systems using immobilized organometalcarbonyl probe groups.

Tricarbonyl(eta 5-cyclohexadienyl)iron(0) and dicarbonyl(triphenylphosphine)(eta 5-cyclo-hexadienyl)iron(0) were derivatized by attachment of an aminopropylsilyl link and covalently attached to fumed silica particles. The fumed silica was coated onto the ZnSe element of an attenuated total reflection (ATR) cell for Fourier transform infrared (FTIR) spectroscopic analysis. The immobilized organometalcarbonyl probe groups are shown to retain their capacity to function as a key element of a molecular sensor assembly and the nu(CO) bands of the two probe groups were interrogated to calibrate the responses for 0-5% levels of dodecane in cyclohexanol to within +/- 0.1%. The potential for dual sensing is described and the simultaneous monitoring of two discrete regions of a dynamically varying inhomogeneous system is reported for the determination of dodecane in cyclohexanol as solutions mix across a permeable barrier in the ATR cell.

Journal Article↗
PubMed2002

O2 plasma treated polyimide-based humidity sensors.

The effect of non-plasma and plasma treated polyimide-based humidity sensors is presented. Pure oxygen was used to etch polyimide in a plasma etcher. The sensor treated in a plasma exhibited higher sensitivity and faster response speed against moisture. The plasma treated sensor had 3.4 times the sensitivity and responded almost twice as fast as the non-plasma treated sensor. A further comparison of sensor outputs, sensitivity and response speed are presented. Chemical analysis of the polyimide surface was carried out by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (ATR-FTIR). XPS and ATR-FTIR showed the increase in carbonyl carbon bonds, C=O, after the plasma treatment. Geometrical modification was observed by atomic force microscopy (AFM). It showed considerable surface roughness after the plasma treatment. O2 plasma treatment improved the sensitivity, and reduced the hysteresis of the sensor due to the increase in C=O bonds in the polyimide.

Journal Article↗
PubMed2003

The automated sample preparation system MixMaster for investigation of volatile organic compounds with mid-infrared evanescent wave spectroscopy.

For efficient development assessment, and calibration of new chemical analyzers a large number of independently prepared samples of target analytes is necessary. Whereas mixing units for gas analysis are readily available, there is a lack of instrumentation for accurate preparation of liquid samples containing volatile organic compounds (VOCs). Manual preparation of liquid samples containing VOCs at trace concentration levels is a particularly challenging and time consuming task. Furthermore, regularly scheduled calibration of sensors and analyzer systems demands for computer controlled automated sample preparation systems. In this paper we present a novel liquid mixing device enabling extensive measurement series with focus on volatile organic compounds, facilitating analysis of water polluted by traces of volatile hydrocarbons. After discussing the mixing system and control software, first results obtained by coupling with an FT-IR spectrometer are reported. Properties of the mixing system are assessed by mid-infrared attenuated total reflection (ATR) spectroscopy of methanol-acetone mixtures and by investigation of multicomponent samples containing volatile hydrocarbons such as 1,2,4-trichlorobenzene and tetrachloroethylene. Obtained ATR spectra are evaluated by principal component regression (PCR) algorithms. It is demonstrated that the presented sample mixing device provides reliable multicomponent mixtures with sufficient accuracy and reproducibility at trace concentration levels.

Electronic Data Processing↗
PubMed2004

Comparison of diffuse-reflectance absorbance and attenuated total reflectance FT-IR for the discrimination of bacteria.

A collection of bacteria belonging to the genus Bacillus were analysed by diffuse reflectance absorbance and attenuated total reflectance Fourier transform infrared (FT-IR) spectroscopy in the mid-infrared. The diffuse reflectance absorbance method is a rapid whole organism fingerprinting method, which generates a biochemical profile of the bacteria, where samples are presented to the FT-IR spectrometer dried on a metal carrier. The attenuated total reflectance FT-IR used in conjunction with a diamond attenuated total reflectance (ATR) accessory produces a biochemical profile of the surface chemistry of bacteria directly without the need for drying, and has not previously been used in the discrimination of bacteria. Principal component, discriminant function and hierarchical cluster analysis were performed on the data to discriminate the bacteria. The differentiation of the bacteria to species level was observed in both analyses however, it was concluded that the ATR FT-IR illustrated better sub-species differentiation of the microorganisms. This may imply that the total biochemical profiling infers discrimination to species level whereas strain specific markers are present on the cell surface chemistry.

Bacillus↗
PubMed2006

An FTIR investigation of isocyanate skin absorption using in vitro guinea pig skin.

Isocyanates may cause contact dermatitis, sensitization and asthma. Dermal exposure to aliphatic and aromatic isocyanates can occur in various exposure settings. The fate of isocyanates on skin is an important unanswered question. Do they react and bind to the outer layer of skin or do they penetrate through the epidermis as unreacted compounds? Knowing the kinetics of these processes is important in developing dermal exposure sampling or decontamination strategies, as well as understanding potential health implications such exposure may have. In this paper the residence time of model isocyanates on hairless guinea pig skin was investigated in vitro using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrometry. Model isocyanates tested were octyl isocyanate, polymeric hexamethylene diisocyanate isocyanurate (pHDI), polymeric isophorone diisocyanate isocyanurate (pIPDI) and methylenediphenyl diisocyanate (MDI). Isocyanates in ethyl acetate (30 microL) were spiked directly on the skin to give 0.2-1.8 micromol NCO cm(-2) (NCO = -N=C=O), and absorbance of the isocyanate group and other chemical groups of the molecule were monitored over time. The ATR-FTIR findings showed that polymeric isocyanates pHDI and pIPDI may remain on the skin as unreacted species for many hours, with only 15-20% of the total isocyanate group disappearing in one hour, while smaller compounds octyl isocyanate and MDI rapidly disappear from the skin surface (80+% in 30 min). Isocyanates most likely leave the skin surface by diffusion predominantly, with minimal reaction with surface proteins. The significance of these findings and their implications for dermal exposure sampling and isocyanate skin decontamination are discussed.

Animals↗
PubMed2002

Ionizing radiation induces ataxia telangiectasia mutated kinase (ATM)-mediated phosphorylation of LKB1/STK11 at Thr-366.

The serine/threonine protein kinase LKB1 functions as a tumour suppressor, and mutations in this enzyme lead to the inherited Peutz-Jeghers cancer syndrome. We previously found that LKB1 was phosphorylated at Thr-366 in vivo, a residue conserved in mammalian, Xenopus and Drosophila LKB1, located on a C-terminal non-catalytic moiety of the enzyme. Mutation of Thr-366 to Ala or Asp partially inhibited the ability of LKB1 to suppress growth of G361 melanoma cells, but did not affect LKB1 activity in vitro or LKB1 localization in vivo. As a first step in exploring the role of this phosphorylation further, we have generated a phosphospecific antibody specifically recognizing LKB1 phosphorylated at Thr-366 and demonstrate that exposure of cells to ionizing radiation (IR) induced a marked phosphorylation of LKB1 at Thr-366 in the nucleus. Thr-366 lies in an optimal phosphorylation motif for the phosphoinositide 3-kinase-like kinases DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated kinase (ATM) and ataxia telangiectasia-related kinase (ATR), which function as sensors for DNA damage in cells and mediate cellular responses to DNA damage. We demonstrate that both DNA-PK and ATM efficiently phosphorylate LKB1 at Thr-366 in vitro and provide evidence that ATM mediates this phosphorylation in vivo. This is based on the finding that LKB1 is not phosphorylated in a cell line lacking ATM in response to IR, and that agents which induce cellular responses via ATR in preference to ATM poorly induce phosphorylation of LKB1 at Thr-366. These observations provide the first link between ATM and LKB1 and suggest that ATM could regulate LKB1.

AMP-Activated Protein Kinase Kinases↗
PubMed2005

Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage.

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and checkpoint activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.

Adenocarcinoma↗
PubMedDate not supplied

[Fanconi anemia: genes and function(s) revisited].

Fanconi anemia (FA), a rare inherited disorder, exhibits a complex phenotype including progressive bone marrow failure, congenital malformations and increased risk of cancers, mainly acute myeloid leukaemia. At the cellular level, FA is characterized by hypersensitivity to DNA cross-linking agents and by high frequencies of induced chromosomal aberrations, a property used for diagnosis. FA results from mutations in one of the eleven FANC (FANCA to FANCJ) genes. Nine of them have been identified. In addition, FANCD1 gene has been shown to be identical to BRCA2, one of the two breast cancer susceptibility genes. Seven of the FANC proteins form a complex, which exists in four different forms depending of its subcellular localisation. Four FANC proteins (D1(BRCA2), D2, I and J) are not associated to the complex. The presence of the nuclear form of the FA core complex is necessary for the mono-ubiquitinylation of FANCD2 protein, a modification required for its re-localization to nuclear foci, likely to be sites of DNA repair. A clue towards understanding the molecular function of the FANC genes comes from the recently identified connection of FANC to the BRCA1, ATM, NBS1 and ATR genes. Two of the FANC proteins (A and D2) directly interact with BRCA1, which in turn interacts with the MRE11/RAD50/NBS1 complex, which is one of the key components in the mechanisms involved in the cellular response to DNA double strand breaks (DSB). Moreover, ATM, a protein kinase that plays a central role in the network of DSB signalling, phosphorylates in vitro and in vivo FANCD2 in response to ionising radiations. Moreover, the NBS1 protein and the monoubiquitinated form of FANCD2 seem to act together in response to DNA crosslinking agents. Taken together with the previously reported impaired DSB and DNA interstrand crosslinks repair in FA cells, the connection of FANC genes to the ATM, ATR, NBS1 and BRCA1 links the FANC genes function to the finely orchestrated network involved in the sensing, signalling and repair of DNA replication-blocking lesions.

Chromosome Aberrations↗
PubMed2003

Diagnostic value of onset-recordings and marker annotations in dual chamber pacemaker stored electrograms.

AIMS: Stored electrograms (EGM) have recently been introduced into pacemaker therapy. New generation devices offer the possibility to store the onset of the EGM (several seconds preceding storage trigger) and marker annotations. The aim of the study was to evaluate whether the diagnostic capabilities of EGMs are improved by these new features. METHODS: We studied 65 patients (age 68+/-12 years, 41 male) implanted with a DDDR-system (PulsarMax II 1280; Guidant). During a 1-month period 319 EGMs have been recorded. EGM triggers were: Ventricular Tachycardia (VT), Non-Sustained VT (NSVT), Atrial Tachycardia Response (ATR), Pacemaker Mediated Tachycardia (PMT) and Sudden Bradycardia Response (SBR). First, each EGM was analysed with onset and markers blinded. EGMs were classified with respect to their trigger as confirmed, not-confirmed or false-positive. Analysis was then repeated with markers visible but without onset, and thereafter vice versa. Finally, EGMs were analysed with both features. It was noted whether the presence of marker annotations and/or onset-recording changed the initial classification of the stored EGMs. RESULTS: 169 EGMs were triggered by SBR, which can only be confirmed with onset recording. False positive EGMs (atrial undersensing) occurred in 12%. The remaining 150 EGMs were triggered by ATR (80%), NSVT (11%), VT (6%) and by PMT (3%). Without onset/markers 37 of these 150 EGMs (25%) could not be confirmed. With markers and onset 33/37 (89%) of these EGMs could now be classified as confirmed or false positive. These EGMs became diagnostic with onset alone in 24%, with markers alone in 24%, and with the combination of both in 41%. In 4 EGMs no definite diagnosis could be established. CONCLUSION: Stored electrograms provide direct insights into device function, thereby providing a validation of diagnostic data. The expanded recording of onset and markers results in markedly improved diagnostic capabilities - compared with conventional EGMs. These new features were necessary to interpret correctly 61% of all stored electrograms, without which a diagnosis would not have been possible. Both onset-recording and marker annotation are necessary for optimal analysis.

Aged↗
PubMed1991

Organization of segmental reflexes in trained dancers.

Unilateral and conditioned Achilles tendontap reflex (ATR) characteristics were examined in a group of skilled dancers and a group of sedentary, untrained subjects (n = 7/group). For the conditioned reflex, the right ATR was conditioned by a tap to the left Achilles tendon using intervals of 10, 40, 70, 100, 130, 160 and 190 ms. Peak isometric force, contraction time and half-relaxation time were examined on each trial. The results indicated that the trained dancers exhibited less unilateral isometric force and longer half-relaxation times (p less than 0.05) than the untrained subjects. Moreover, the contralateral conditioning stimulus caused a more marked short-latency facilitation as well as a long-latency inhibition in the reflex force characteristics for the trained dancers when compared with the untrained subjects. These differences in both simple and conditioned reflexes in individuals trained for dance activities may reflect differences in muscle stiffness, tissue compliance or neural organization.

Achilles Tendon↗
PubMed2005

BRIT1/MCPH1 is a DNA damage responsive protein that regulates the Brca1-Chk1 pathway, implicating checkpoint dysfunction in microcephaly.

BRIT1 [BRCT-repeat inhibitor of hTERT expression], a repressor of human telomerase function, is implicated in cellular immortalization. Here, we find that BRIT1 acts as a regulator of both the intra-S and G2/M checkpoints. When BRIT1 expression is depleted, cells lose the ionizing radiation (IR)-induced cell cycle arrest and become IR sensitive. BRIT1 is a chromatin-associated protein that forms irradiation-induced nuclear foci that colocalize with gamma-H2AX foci. BRIT1 is also required for the expression of both BRCA1 and the checkpoint kinase Chk1 and phosphorylation of Nbs1. Thus, the checkpoint defects in the absence of BRIT1 are likely to result from its regulation of Nbs1, BRCA1, and Chk1. BRIT1 is identical to the recently discovered MCPH1 gene, found mutant in patients with primary microcephaly. The ataxia telangiectasia mutated-Rad3 related (ATR)-Chk1 pathway is defective in Seckel syndrome, another microcephaly disorder. We propose that the microcephaly observed in patients with MCPH1 deficiencies is due to disruption of the ATR-BRCA1-Chk1 signaling pathway that is also disrupted in Seckel syndrome patients.

BRCA1 Protein↗
PubMed2001

ATM phosphorylates histone H2AX in response to DNA double-strand breaks.

A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ATM and Rad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs-/- and Atm-/- cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs-/- cells and severely compromised in Atm-/- cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm-/- fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm-/- fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks.

Androstadienes↗
PubMed2003

Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes.

DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX (gammaH2AX) foci. Here we show that gammaH2AX formation is also a cellular response to topoisomerase I cleavage complexes known to induce DNA double-strand breaks during replication. In HCT116 human carcinoma cells exposed to the topoisomerase I inhibitor camptothecin, the resulting gammaH2AX formation can be prevented with the phosphatidylinositol 3-OH kinase-related kinase inhibitor wortmannin; however, in contrast to ionizing radiation, only camptothecin-induced gammaH2AX formation can be prevented with the DNA replication inhibitor aphidicolin and enhanced with the checkpoint abrogator 7-hydroxystaurosporine. This gammaH2AX formation is suppressed in ATR (ataxia telangiectasia and Rad3-related) deficient cells and markedly decreased in DNA-dependent protein kinase-deficient cells but is not abrogated in ataxia telangiectasia cells, indicating that ATR and DNA-dependent protein kinase are the kinases primarily involved in gammaH2AX formation at the sites of replication-mediated DNA double-strand breaks. Mre11- and Nbs1-deficient cells are still able to form gammaH2AX. However, H2AX-/- mouse embryonic fibroblasts exposed to camptothecin fail to form Mre11, Rad50, and Nbs1 foci and are hypersensitive to camptothecin. These results demonstrate a conserved gammaH2AX response for double-strand breaks induced by replication fork collision. gammaH2AX foci are required for recruiting repair and checkpoint protein complexes to the replication break sites.

Animals↗