Emmy Rogakou - Academia.edu (original) (raw)

Papers by Emmy Rogakou

La presente invention concerne un procede et un kit permettant de determiner les ruptures a doubl... more La presente invention concerne un procede et un kit permettant de determiner les ruptures a doubles brins dans d'ADN. Ce procede consiste a mettre en contact un echantillon comprenant des proteines d'histone H2A avec l'anticorps purifie ou isole ou un fragment de ce dernier presentant une reaction antigenique qui se lie, de maniere specifique, a la serine phosphorylee a terminaison C dans une proteine d'histone K2A. Ce procede consiste ensuite a detecter la liaison de l'anticorps ou du fragment de ce dernier a une proteine d'histone H2A dans l'echantillon. La detection de la liaison de l'anticorps ou du fragment de ce dernier a la proteine d'histone H2A indique la presence d'une rupture a double brins d'ADN dans l'ADN.

Current Biology, 2000

Background: The response of eukaryotic cells to double-strand breaks in genomic DNA includes the ... more Background: The response of eukaryotic cells to double-strand breaks in genomic DNA includes the sequestration of many factors into nuclear foci. Recently it has been reported that a member of the histone H2A family, H2AX, becomes extensively phosphorylated within 1-3 minutes of DNA damage and forms foci at break sites. Results: In this work, we examine the role of H2AX phosphorylation in focus formation by several repair-related complexes, and investigate what factors may be involved in initiating this response. Using two different methods to create DNA double-strand breaks in human cells, we found that the repair factors Rad50 and Rad51 each colocalized with phosphorylated H2AX (γ-H2AX) foci after DNA damage. The product of the tumor suppressor gene BRCA1 also colocalized with γ-H2AX and was recruited to these sites before Rad50 or Rad51. Exposure of cells to the fungal inhibitor wortmannin eliminated focus formation by all repair factors examined, suggesting a role for the phosphoinositide (PI)-3 family of protein kinases in mediating this response. Wortmannin treatment was effective only when it was added early enough to prevent γ-H2AX formation, indicating that γ-H2AX is necessary for the recruitment of other factors to the sites of DNA damage. DNA repair-deficient cells exhibit a substantially reduced ability to increase the phosphorylation of H2AX in response to ionizing radiation, consistent with a role for γ-H2AX in DNA repair. Conclusions: The pattern of γ-H2AX foci that is established within a few minutes of DNA damage accounts for the patterns of Rad50, Rad51, and Brca1 foci seen much later during recovery from damage. The evidence presented strongly supports a role for the γ-H2AX and the PI-3 protein kinase family in focus formation at sites of double-strand breaks and suggests the possibility of a change in chromatin structure accompanying double-strand break repair.

International Journal of Molecular Sciences

γH2AX has emerged in the last 20 years as a central player in the DDR (DNA damage response), with... more γH2AX has emerged in the last 20 years as a central player in the DDR (DNA damage response), with specificity for DSBs (double-strand breaks). Upon the generation of DSBs, γ-phosphorylation extends along megabase-long domains in chromatin, both sides of the damage. The significance of this mechanism is of great importance; it depicts a biological amplification mechanism where one DSB induces the γ-phosphorylation of thousands of H2AX molecules along megabaselong domains of chromatin, that are adjusted to the sites of DSBs. A sequential recruitment of signal transduction factors that interact to each other and become activated to further amplify the signal that will travel to the cytoplasm take place on the γ-phosphorylated chromatin. γ-phosphorylation is an early event in the DSB damage response, induced in all phases of the cell cycle, and participates in both DSB repair pathways, the HR (homologous recombination) and NHEJ (non-homologous end joining). Today, numerous studies support the notion that γH2AX functions as a guardian of the genome by preventing misrepaired DSB that increase the mutation load of the cells and may further lead to genome instability and carcinogenesis.

Cancers, Jan 18, 2017

Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly d... more Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs), single strand breaks (SSBs) and base lesions within a short DNA region of up to 15-20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1) repair resistant, increasing genomic instability (GI) and malignant transformation and (2) can be considered as pe...

Radiat Res, 2002

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into ... more When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (␥-H2AX) demonstrates that ␥-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of 125 I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of 125 IdU and processed immunocytochemically to determine the number of ␥-H2AX foci. The numbers of 125 IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of 125 I decays and the number of foci per cell, consistent with the assumptions that each 125 I decay yields a DNA DSB and each DNA DSB yields a visible ␥-H2AX focus. Based on these findings, we conclude that ␥-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.

Radiation Research, 2002

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into ... more When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (␥-H2AX) demonstrates that ␥-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of 125 I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of 125 IdU and processed immunocytochemically to determine the number of ␥-H2AX foci. The numbers of 125 IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of 125 I decays and the number of foci per cell, consistent with the assumptions that each 125 I decay yields a DNA DSB and each DNA DSB yields a visible ␥-H2AX focus. Based on these findings, we conclude that ␥-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.

Epigenetics, the Environment, and Children’s Health Across Lifespans, 2016

Cancer Drug Discovery and Development, 2007

One of the most fascinating themes in the biology of double-strand breaks (DSBs) is that chromati... more One of the most fascinating themes in the biology of double-strand breaks (DSBs) is that chromatin is emerging as a multifunctional player in the DSB damage response. The phosphorylation of H2AX on Ser 139, named γH2AX, is an early response to the generation of DNA DSBs and extends along megabase-long domains, both sites of the lesion, supporting amplification of signal transduction pathways. In parallel, 53BP1 accumulates on damaged chromatin to interface between methylated histone residues and proteins that belong to the signal-transduction pathways, mediating cell-cycle arrest or apoptosis. Interestingly, the two pathways crosstalk at the chromatin level.

DNA Damage Recognition, 2005

Nature genetics, 2001

In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand ... more In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand breaks (DSBs), a process that precedes homologous synapsis. Here we use an antibody specific for a phosphorylated histone (gamma-H2AX, which marks the sites of DSBs) to investigate the timing, distribution and Spo11-dependence of meiotic DSBs in the mouse. We show that, as in yeast, recombination in the mouse is initiated by Spo11-dependent DSBs that form during leptotene. Loss of gamma-H2AX staining (which in irradiated somatic cells is temporally linked with DSB repair) is temporally and spatially correlated with synapsis, even when this synapsis is 'non-homologous'.

Nature Genetics, 2001

In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand ... more In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand breaks (DSBs), a process that precedes homologous synapsis. Here we use an antibody specific for a phosphorylated histone (-H2AX, which marks the sites of DSBs) to investigate the timing, distribution and Spo11-dependence of meiotic DSBs in the mouse. We show that, as in yeast, recombination in the mouse is initiated

Science, 2000

SDS-polyacrylamide gel electrophoresis. After gel separation, proteins were electroblotted onto n... more SDS-polyacrylamide gel electrophoresis. After gel separation, proteins were electroblotted onto nitrocellulose membranes and incubated with polyclonal antibodies that recognize PC1/3 and PC2 (provided by I. Lindberg, Louisiana State Medical Center). Membranes were washed and then incubated with goat antiserum to rabbit coupled to horseradish peroxidase (Amersham Pharmacia Biotech, Uppsala, Sweden). The blots were then developed with a chemiluminescence Western blotting detection kit. 23. GTC-1 cells grown to 70 to 80% confluence in 12-well plates were given restricted nutrients for 2 hours in Dulbecco's minimum essential medium (DMEM) with 1.0 mM glucose and 1% fetal calf serum (FCS). Cells were washed and then incubated in 0.5 ml of release media (DMEM plus 1% FCS with either 1.0 or 10.0 mM of glucose) for 2 hours. Insulin levels in media were measured using the human-specific insulin ELISA kit [American Laboratory Products Company (ALPCO), Windham, NH] according to supplier's instructions. 24. The GIP/Ins fragment (4.2 kb) was excised with Hind III and gel-purified. Transgenic mice were generated by pronuclear microinjection of the purified transgene into fertilized embryos that were then implanted into pseudopregnant females. Transgenic mice were identified by Southern blot analysis. Ear sections were digested, and the purified DNA was cut with Xho I and Pvu II (Fig. 1C), electrophoretically separated, and transferred to nylon membrane. For the detection of the transgene, a 416bp human insulin gene fragment encompassing intron 2 was amplified by using primers 2 and 4 (Fig. 1C). The PCR product was prepared as a probe by radiolabeling with [␣-32 P]dCTP, and bands were detected by autoradiography. Southern analysis results were further confirmed by PCR amplification of the genomic DNA using primers 2 and 4. Positive founders were outbred with wild-type FVB/N mice to establish transgenic lines. 25. Primers used were human proinsulin-specific, forward 5Ј-CCAGCCGCAGCCT T TGTGA-3Ј and reverse 5Ј-GGTACAGCAT TGT TCCACAATG-3Ј; mouse proinsulin-specific, forward 5Ј-ACCACCAGCCCTAAGT-GAT-3Ј and reverse 5Ј-CTAGT TGCAGTAGT TCTC-CAGC-3Ј. PCR conditions were as follows: denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min for 45 cycles. PCR products were analyzed on a 2% agarose gel and visualized by ethidium bromide staining. The humanand mouse-specific primer sets yield 350-bp and 396-bp products, respectively. 26. Tissues were fixed in Bouin's solution overnight and embedded in paraffin. Tissue sections 5 m thick were mounted on glass slides. For immunohistochemistry, the avidin-biotin complex method was used with peroxidase and diaminobenzidine as the chromogen. Sections were incubated with guinea pig antibody to insulin (1: 500, Linco Research, St. Charles, MO) or mouse antibody to GIP (1: 200, a gift from R. Pederson, University of British Columbia) for 30 min and appropriate secondary antibodies for 20 min at room temperature. Biotinylated secondary antibodies were used for immunohistochemistry, and fluorescein-or Cy3conjugated secondary antibodies were used for immunofluorescence. 27. Plasma insulin levels were measured using the ultrasensitive human-specific insulin ELISA kit (ALPCO) according to supplier's instructions. This assay has Ͻ0.01% cross-reactivity with human proinsulin and C peptide and does not detect mouse insulin. Plasma C-peptide measurements were made with a rat/ mouse C-peptide radioimmunoassay kit (Linco Research). The assay displays no cross-reactivity with human C peptide. 28. Streptozotocin was administered to 8-week-old transgenic and age-matched control mice via an intraperitoneal injection at a dose of 200 mg/kg body weight in citrate buffer. At this high dose of streptozotocin, mice typically display glucosuria within 3 days after injection. For oral glucose tolerance tests, glucose was administered orally by feeding tube (1.5 g/kg body weight) as a 50% solution (w/v) to mice that had been without food for 14 hours. Blood samples (40 l) were collected from the tail vein of conscious mice at 0, 10, 20, 30, 60, 90, and 120 min after the glucose load. Plasma glucose levels were determined by enzymatic, colorimetric assay (Sigma), and plasma insulin levels were measured using the ultrasensitive human-specific insulin ELISA kit (27). 29. Pancreata were homogenized and then sonicated at 4°C in 2 mM acetic acid containing 0.25% bovine serum albumin. After incubation for 2 hours on ice, tissue homogenates were resonicated and centrifuged (8000g, 20 min), and supernatants were assayed for insulin by radioimmunoassay.

Radiation Research, 2002

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into ... more When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (␥-H2AX) demonstrates that ␥-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of 125 I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of 125 IdU and processed immunocytochemically to determine the number of ␥-H2AX foci. The numbers of 125 IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of 125 I decays and the number of foci per cell, consistent with the assumptions that each 125 I decay yields a DNA DSB and each DNA DSB yields a visible ␥-H2AX focus. Based on these findings, we conclude that ␥-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.

Journal of Biological Chemistry, 2004

The histone variant H2AX is rapidly phosphorylated (denoted ␥H2AX) in large chromatin domains (fo... more The histone variant H2AX is rapidly phosphorylated (denoted ␥H2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for ␥H2AX in DNA repair; however, ␥H2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on ␥H2AX for anchoring free broken ends versus ends that are held together during programmed breakage-joining reactions. Retroviral DNA integration produces a unique intermediate in which a dsDNA break in host DNA is held together by the intervening viral DNA, and such a reaction provides a useful model to distinguish ␥H2AX functions. We found that integration promotes transient formation of ␥H2AX at retroviral integration sites as detected by both immunocytological and chromatin immunoprecipitation methods. These results provide the first direct evidence for the association of newly integrated viral DNA with a protein species that is an established marker for the onset of a DNA damage response. We also show that H2AX is not required for repair of the retroviral integration intermediate as determined by stable transduction. These observations provide independent support for an anchoring model for the function of ␥H2AX in chromatin repair.

Journal of Biological Chemistry, 2000

Histone H2AX is a ubiquitous member of the H2A histone family that differs from the other H2A his... more Histone H2AX is a ubiquitous member of the H2A histone family that differs from the other H2A histones by the presence of an evolutionarily conserved C-terminal motif,-KKATQASQEY. The serine residue in this motif becomes rapidly phosphorylated in cells and animals when DNA double-stranded breaks are introduced into their chromatin by various physical and chemical means. In the present communication we show that this phosphorylated form of H2AX, referred to as ␥-H2AX, appears during apoptosis concurrently with the initial appearance of high molecular weight DNA fragments. ␥-H2AX forms before the appearance of internucleosomal DNA fragments and the externalization of phosphatidylserine to the outer membrane leaflet. ␥-H2AX formation is inhibited by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and the inhibitor of caspaseactivated DNase, and it is induced when DNase I and restriction enzymes are introduced into cells, suggesting that any apoptotic endonuclease is sufficient to induce ␥-H2AX formation. These results indicate that ␥-H2AX formation is an early chromatin modification following initiation of DNA fragmentation during apoptosis.

Journal of Biological Chemistry, 1998

When mammalian cell cultures or mice are exposed to ionizing radiation in survivable or lethal am... more When mammalian cell cultures or mice are exposed to ionizing radiation in survivable or lethal amounts, novel mass components are found in the histone H2A region of two-dimensional gels. Collectively referred to as ␥, these components are formed in vivo by several procedures that introduce double-stranded breaks into DNA. ␥-Components, which appeared to be the only major novel components detected by mass or 32 PO 4 incorporation on acetic acid-urea-Triton X-100-acetic acid-ureacetyltrimethylammonium bromide or SDS-acetic acidurea-cetyltrimethylammonium bromide gels after exposure of cells to ionizing radiation, are shown to be histone H2AX species that have been phosphorylated specifically at serine 139. ␥-H2AX appears rapidly after exposure of cell cultures to ionizing radiation; half-maximal amounts are reached by 1 min and maximal amounts by 10 min. At the maximum, approximately 1% of the H2AX becomes ␥-phosphorylated per gray of ionizing radiation, a finding that indicates that 35 DNA double-stranded breaks, the number introduced by each gray into the 6 ؋ 10 9 base pairs of a mammalian G 1 genome, leads to the ␥-phosphorylation of H2AX distributed over 1% of the chromatin. Thus, about 0.03% of the chromatin appears to be involved per DNA doublestranded break. This value, which corresponds to about 2 ؋ 10 6 base pairs of DNA per double-stranded break, indicates that large amounts of chromatin are involved with each DNA double-stranded break. Thus, ␥-H2AX formation is a rapid and sensitive cellular response to the presence of DNA double-stranded breaks, a response that may provide insight into higher order chromatin structures.

Journal of Biological Chemistry, 2003

DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by t... more DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX (␥H2AX) foci. Here we show that ␥H2AX 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 ␥H2AX formation can be prevented with the phosphatidylinositol 3-OH kinase-related kinase inhibitor wortmannin; however, in contrast to ionizing radiation, only camptothecin-induced ␥H2AX formation can be prevented with the DNA replication inhibitor aphidicolin and enhanced with the checkpoint abrogator 7-hydroxystaurosporine. This ␥H2AX 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 ␥H2AX formation at the sites of replication-mediated DNA doublestrand breaks. Mre11-and Nbs1-deficient cells are still able to form ␥H2AX. 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 ␥H2AX response for double-strand breaks induced by replication fork collision. ␥H2AX foci are required for recruiting repair and checkpoint protein complexes to the replication break sites.

La presente invention concerne un procede et un kit permettant de determiner les ruptures a doubl... more La presente invention concerne un procede et un kit permettant de determiner les ruptures a doubles brins dans d'ADN. Ce procede consiste a mettre en contact un echantillon comprenant des proteines d'histone H2A avec l'anticorps purifie ou isole ou un fragment de ce dernier presentant une reaction antigenique qui se lie, de maniere specifique, a la serine phosphorylee a terminaison C dans une proteine d'histone K2A. Ce procede consiste ensuite a detecter la liaison de l'anticorps ou du fragment de ce dernier a une proteine d'histone H2A dans l'echantillon. La detection de la liaison de l'anticorps ou du fragment de ce dernier a la proteine d'histone H2A indique la presence d'une rupture a double brins d'ADN dans l'ADN.

Current Biology, 2000

Background: The response of eukaryotic cells to double-strand breaks in genomic DNA includes the ... more Background: The response of eukaryotic cells to double-strand breaks in genomic DNA includes the sequestration of many factors into nuclear foci. Recently it has been reported that a member of the histone H2A family, H2AX, becomes extensively phosphorylated within 1-3 minutes of DNA damage and forms foci at break sites. Results: In this work, we examine the role of H2AX phosphorylation in focus formation by several repair-related complexes, and investigate what factors may be involved in initiating this response. Using two different methods to create DNA double-strand breaks in human cells, we found that the repair factors Rad50 and Rad51 each colocalized with phosphorylated H2AX (γ-H2AX) foci after DNA damage. The product of the tumor suppressor gene BRCA1 also colocalized with γ-H2AX and was recruited to these sites before Rad50 or Rad51. Exposure of cells to the fungal inhibitor wortmannin eliminated focus formation by all repair factors examined, suggesting a role for the phosphoinositide (PI)-3 family of protein kinases in mediating this response. Wortmannin treatment was effective only when it was added early enough to prevent γ-H2AX formation, indicating that γ-H2AX is necessary for the recruitment of other factors to the sites of DNA damage. DNA repair-deficient cells exhibit a substantially reduced ability to increase the phosphorylation of H2AX in response to ionizing radiation, consistent with a role for γ-H2AX in DNA repair. Conclusions: The pattern of γ-H2AX foci that is established within a few minutes of DNA damage accounts for the patterns of Rad50, Rad51, and Brca1 foci seen much later during recovery from damage. The evidence presented strongly supports a role for the γ-H2AX and the PI-3 protein kinase family in focus formation at sites of double-strand breaks and suggests the possibility of a change in chromatin structure accompanying double-strand break repair.

International Journal of Molecular Sciences

γH2AX has emerged in the last 20 years as a central player in the DDR (DNA damage response), with... more γH2AX has emerged in the last 20 years as a central player in the DDR (DNA damage response), with specificity for DSBs (double-strand breaks). Upon the generation of DSBs, γ-phosphorylation extends along megabase-long domains in chromatin, both sides of the damage. The significance of this mechanism is of great importance; it depicts a biological amplification mechanism where one DSB induces the γ-phosphorylation of thousands of H2AX molecules along megabaselong domains of chromatin, that are adjusted to the sites of DSBs. A sequential recruitment of signal transduction factors that interact to each other and become activated to further amplify the signal that will travel to the cytoplasm take place on the γ-phosphorylated chromatin. γ-phosphorylation is an early event in the DSB damage response, induced in all phases of the cell cycle, and participates in both DSB repair pathways, the HR (homologous recombination) and NHEJ (non-homologous end joining). Today, numerous studies support the notion that γH2AX functions as a guardian of the genome by preventing misrepaired DSB that increase the mutation load of the cells and may further lead to genome instability and carcinogenesis.

Cancers, Jan 18, 2017

Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly d... more Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs), single strand breaks (SSBs) and base lesions within a short DNA region of up to 15-20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1) repair resistant, increasing genomic instability (GI) and malignant transformation and (2) can be considered as pe...

Radiat Res, 2002

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into ... more When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (␥-H2AX) demonstrates that ␥-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of 125 I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of 125 IdU and processed immunocytochemically to determine the number of ␥-H2AX foci. The numbers of 125 IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of 125 I decays and the number of foci per cell, consistent with the assumptions that each 125 I decay yields a DNA DSB and each DNA DSB yields a visible ␥-H2AX focus. Based on these findings, we conclude that ␥-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.

Radiation Research, 2002

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into ... more When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (␥-H2AX) demonstrates that ␥-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of 125 I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of 125 IdU and processed immunocytochemically to determine the number of ␥-H2AX foci. The numbers of 125 IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of 125 I decays and the number of foci per cell, consistent with the assumptions that each 125 I decay yields a DNA DSB and each DNA DSB yields a visible ␥-H2AX focus. Based on these findings, we conclude that ␥-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.

Epigenetics, the Environment, and Children’s Health Across Lifespans, 2016

Cancer Drug Discovery and Development, 2007

One of the most fascinating themes in the biology of double-strand breaks (DSBs) is that chromati... more One of the most fascinating themes in the biology of double-strand breaks (DSBs) is that chromatin is emerging as a multifunctional player in the DSB damage response. The phosphorylation of H2AX on Ser 139, named γH2AX, is an early response to the generation of DNA DSBs and extends along megabase-long domains, both sites of the lesion, supporting amplification of signal transduction pathways. In parallel, 53BP1 accumulates on damaged chromatin to interface between methylated histone residues and proteins that belong to the signal-transduction pathways, mediating cell-cycle arrest or apoptosis. Interestingly, the two pathways crosstalk at the chromatin level.

DNA Damage Recognition, 2005

Nature genetics, 2001

In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand ... more In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand breaks (DSBs), a process that precedes homologous synapsis. Here we use an antibody specific for a phosphorylated histone (gamma-H2AX, which marks the sites of DSBs) to investigate the timing, distribution and Spo11-dependence of meiotic DSBs in the mouse. We show that, as in yeast, recombination in the mouse is initiated by Spo11-dependent DSBs that form during leptotene. Loss of gamma-H2AX staining (which in irradiated somatic cells is temporally linked with DSB repair) is temporally and spatially correlated with synapsis, even when this synapsis is 'non-homologous'.

Nature Genetics, 2001

In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand ... more In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand breaks (DSBs), a process that precedes homologous synapsis. Here we use an antibody specific for a phosphorylated histone (-H2AX, which marks the sites of DSBs) to investigate the timing, distribution and Spo11-dependence of meiotic DSBs in the mouse. We show that, as in yeast, recombination in the mouse is initiated

Science, 2000

SDS-polyacrylamide gel electrophoresis. After gel separation, proteins were electroblotted onto n... more SDS-polyacrylamide gel electrophoresis. After gel separation, proteins were electroblotted onto nitrocellulose membranes and incubated with polyclonal antibodies that recognize PC1/3 and PC2 (provided by I. Lindberg, Louisiana State Medical Center). Membranes were washed and then incubated with goat antiserum to rabbit coupled to horseradish peroxidase (Amersham Pharmacia Biotech, Uppsala, Sweden). The blots were then developed with a chemiluminescence Western blotting detection kit. 23. GTC-1 cells grown to 70 to 80% confluence in 12-well plates were given restricted nutrients for 2 hours in Dulbecco's minimum essential medium (DMEM) with 1.0 mM glucose and 1% fetal calf serum (FCS). Cells were washed and then incubated in 0.5 ml of release media (DMEM plus 1% FCS with either 1.0 or 10.0 mM of glucose) for 2 hours. Insulin levels in media were measured using the human-specific insulin ELISA kit [American Laboratory Products Company (ALPCO), Windham, NH] according to supplier's instructions. 24. The GIP/Ins fragment (4.2 kb) was excised with Hind III and gel-purified. Transgenic mice were generated by pronuclear microinjection of the purified transgene into fertilized embryos that were then implanted into pseudopregnant females. Transgenic mice were identified by Southern blot analysis. Ear sections were digested, and the purified DNA was cut with Xho I and Pvu II (Fig. 1C), electrophoretically separated, and transferred to nylon membrane. For the detection of the transgene, a 416bp human insulin gene fragment encompassing intron 2 was amplified by using primers 2 and 4 (Fig. 1C). The PCR product was prepared as a probe by radiolabeling with [␣-32 P]dCTP, and bands were detected by autoradiography. Southern analysis results were further confirmed by PCR amplification of the genomic DNA using primers 2 and 4. Positive founders were outbred with wild-type FVB/N mice to establish transgenic lines. 25. Primers used were human proinsulin-specific, forward 5Ј-CCAGCCGCAGCCT T TGTGA-3Ј and reverse 5Ј-GGTACAGCAT TGT TCCACAATG-3Ј; mouse proinsulin-specific, forward 5Ј-ACCACCAGCCCTAAGT-GAT-3Ј and reverse 5Ј-CTAGT TGCAGTAGT TCTC-CAGC-3Ј. PCR conditions were as follows: denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min for 45 cycles. PCR products were analyzed on a 2% agarose gel and visualized by ethidium bromide staining. The humanand mouse-specific primer sets yield 350-bp and 396-bp products, respectively. 26. Tissues were fixed in Bouin's solution overnight and embedded in paraffin. Tissue sections 5 m thick were mounted on glass slides. For immunohistochemistry, the avidin-biotin complex method was used with peroxidase and diaminobenzidine as the chromogen. Sections were incubated with guinea pig antibody to insulin (1: 500, Linco Research, St. Charles, MO) or mouse antibody to GIP (1: 200, a gift from R. Pederson, University of British Columbia) for 30 min and appropriate secondary antibodies for 20 min at room temperature. Biotinylated secondary antibodies were used for immunohistochemistry, and fluorescein-or Cy3conjugated secondary antibodies were used for immunofluorescence. 27. Plasma insulin levels were measured using the ultrasensitive human-specific insulin ELISA kit (ALPCO) according to supplier's instructions. This assay has Ͻ0.01% cross-reactivity with human proinsulin and C peptide and does not detect mouse insulin. Plasma C-peptide measurements were made with a rat/ mouse C-peptide radioimmunoassay kit (Linco Research). The assay displays no cross-reactivity with human C peptide. 28. Streptozotocin was administered to 8-week-old transgenic and age-matched control mice via an intraperitoneal injection at a dose of 200 mg/kg body weight in citrate buffer. At this high dose of streptozotocin, mice typically display glucosuria within 3 days after injection. For oral glucose tolerance tests, glucose was administered orally by feeding tube (1.5 g/kg body weight) as a 50% solution (w/v) to mice that had been without food for 14 hours. Blood samples (40 l) were collected from the tail vein of conscious mice at 0, 10, 20, 30, 60, 90, and 120 min after the glucose load. Plasma glucose levels were determined by enzymatic, colorimetric assay (Sigma), and plasma insulin levels were measured using the ultrasensitive human-specific insulin ELISA kit (27). 29. Pancreata were homogenized and then sonicated at 4°C in 2 mM acetic acid containing 0.25% bovine serum albumin. After incubation for 2 hours on ice, tissue homogenates were resonicated and centrifuged (8000g, 20 min), and supernatants were assayed for insulin by radioimmunoassay.

Radiation Research, 2002

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into ... more When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (␥-H2AX) demonstrates that ␥-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of 125 I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of 125 IdU and processed immunocytochemically to determine the number of ␥-H2AX foci. The numbers of 125 IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of 125 I decays and the number of foci per cell, consistent with the assumptions that each 125 I decay yields a DNA DSB and each DNA DSB yields a visible ␥-H2AX focus. Based on these findings, we conclude that ␥-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.

Journal of Biological Chemistry, 2004

The histone variant H2AX is rapidly phosphorylated (denoted ␥H2AX) in large chromatin domains (fo... more The histone variant H2AX is rapidly phosphorylated (denoted ␥H2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for ␥H2AX in DNA repair; however, ␥H2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on ␥H2AX for anchoring free broken ends versus ends that are held together during programmed breakage-joining reactions. Retroviral DNA integration produces a unique intermediate in which a dsDNA break in host DNA is held together by the intervening viral DNA, and such a reaction provides a useful model to distinguish ␥H2AX functions. We found that integration promotes transient formation of ␥H2AX at retroviral integration sites as detected by both immunocytological and chromatin immunoprecipitation methods. These results provide the first direct evidence for the association of newly integrated viral DNA with a protein species that is an established marker for the onset of a DNA damage response. We also show that H2AX is not required for repair of the retroviral integration intermediate as determined by stable transduction. These observations provide independent support for an anchoring model for the function of ␥H2AX in chromatin repair.

Journal of Biological Chemistry, 2000

Histone H2AX is a ubiquitous member of the H2A histone family that differs from the other H2A his... more Histone H2AX is a ubiquitous member of the H2A histone family that differs from the other H2A histones by the presence of an evolutionarily conserved C-terminal motif,-KKATQASQEY. The serine residue in this motif becomes rapidly phosphorylated in cells and animals when DNA double-stranded breaks are introduced into their chromatin by various physical and chemical means. In the present communication we show that this phosphorylated form of H2AX, referred to as ␥-H2AX, appears during apoptosis concurrently with the initial appearance of high molecular weight DNA fragments. ␥-H2AX forms before the appearance of internucleosomal DNA fragments and the externalization of phosphatidylserine to the outer membrane leaflet. ␥-H2AX formation is inhibited by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and the inhibitor of caspaseactivated DNase, and it is induced when DNase I and restriction enzymes are introduced into cells, suggesting that any apoptotic endonuclease is sufficient to induce ␥-H2AX formation. These results indicate that ␥-H2AX formation is an early chromatin modification following initiation of DNA fragmentation during apoptosis.

Journal of Biological Chemistry, 1998

When mammalian cell cultures or mice are exposed to ionizing radiation in survivable or lethal am... more When mammalian cell cultures or mice are exposed to ionizing radiation in survivable or lethal amounts, novel mass components are found in the histone H2A region of two-dimensional gels. Collectively referred to as ␥, these components are formed in vivo by several procedures that introduce double-stranded breaks into DNA. ␥-Components, which appeared to be the only major novel components detected by mass or 32 PO 4 incorporation on acetic acid-urea-Triton X-100-acetic acid-ureacetyltrimethylammonium bromide or SDS-acetic acidurea-cetyltrimethylammonium bromide gels after exposure of cells to ionizing radiation, are shown to be histone H2AX species that have been phosphorylated specifically at serine 139. ␥-H2AX appears rapidly after exposure of cell cultures to ionizing radiation; half-maximal amounts are reached by 1 min and maximal amounts by 10 min. At the maximum, approximately 1% of the H2AX becomes ␥-phosphorylated per gray of ionizing radiation, a finding that indicates that 35 DNA double-stranded breaks, the number introduced by each gray into the 6 ؋ 10 9 base pairs of a mammalian G 1 genome, leads to the ␥-phosphorylation of H2AX distributed over 1% of the chromatin. Thus, about 0.03% of the chromatin appears to be involved per DNA doublestranded break. This value, which corresponds to about 2 ؋ 10 6 base pairs of DNA per double-stranded break, indicates that large amounts of chromatin are involved with each DNA double-stranded break. Thus, ␥-H2AX formation is a rapid and sensitive cellular response to the presence of DNA double-stranded breaks, a response that may provide insight into higher order chromatin structures.

Journal of Biological Chemistry, 2003

DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by t... more DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX (␥H2AX) foci. Here we show that ␥H2AX 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 ␥H2AX formation can be prevented with the phosphatidylinositol 3-OH kinase-related kinase inhibitor wortmannin; however, in contrast to ionizing radiation, only camptothecin-induced ␥H2AX formation can be prevented with the DNA replication inhibitor aphidicolin and enhanced with the checkpoint abrogator 7-hydroxystaurosporine. This ␥H2AX 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 ␥H2AX formation at the sites of replication-mediated DNA doublestrand breaks. Mre11-and Nbs1-deficient cells are still able to form ␥H2AX. 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 ␥H2AX response for double-strand breaks induced by replication fork collision. ␥H2AX foci are required for recruiting repair and checkpoint protein complexes to the replication break sites.