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W M Bonner

Publications and source records attributed to W M Bonner.

At least 19 recordsLinked to original sources
PubMed2006

A microbeam study of DNA double-strand breaks in bystander primary human fibroblasts.

Radiation-induced bystander effect has been well documented. However, the mechanisms are poorly understood. How we incorporate this effect into the classical models of risk assessment remains an open question. Here, the induction of bystander effect was studied by assessing DNA double-strand break (DSB) formation in situ with the rapid and sensitive gamma-H2AX focus formation assay. Utilising the Columbia University single-cell microbeam system to deliver 2 or 20 individual alpha particles to selected cell nuclei in a precisely known proportion of cells in a population, the induced DNA DSB incidences were quantified 30 min and 18 h post-IR. The increase in DNA DSB incidence in bystander cells lacked of a linear dose response indicating that neither the dose of irradiation nor proportion of irradiated cells in a population, is a critical parameter. This study confirms a binary all-or-nothing model of triggering the bystander response. The delay and persistence of the bystander response suggests a different mechanism of DSB induction in bystander cells than in directly irradiated cells.

Alpha Particles↗
PubMed2006

p21CDKN1A allows the repair of replication-mediated DNA double-strand breaks induced by topoisomerase I and is inactivated by the checkpoint kinase inhibitor 7-hydroxystaurosporine.

This study provides evidence for the importance of p21(CDKN1A) for the repair of replication-mediated DNA double-strand breaks (DSBs) induced by topoisomerase I. We report that defects of p21(CDKN1A) and p53 enhance camptothecin-induced histone H2AX phosphorylation (gammaH2AX), a marker for DNA DSBs. In human colon carcinoma HCT116 cells with wild-type (wt) p53, gammaH2AX reverses after camptothecin removal. By contrast, gammaH2AX increases after camptothecin removal in HCT116 cells deficient for p53 (p53-/-) or p21(CDKN1A) (p21-/-) as the cells reach the late-S and G2 phases. Since p21-/- cells exhibit similar S-phase arrest as wt cells in response to camptothecin and aphidicolin does not abrogate the enhanced gammaH2AX formation in p21-/- cells, we conclude that enhanced gammaH2AX formation in p21-/- cells is not due to re-replication. The cell cycle checkpoint abrogator and Chk1/Chk2 inhibitor 7-hydroxystaurosporine (UCN-01) also increases camptothecin-induced gammaH2AX formation and inhibits camptothecin-induced p21(CDKN1A) upregulation in HCT116 wt cells. TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) assays demonstrate that gammaH2AX formation in late S and G2 cells following CPT treatment corresponds to DNA breaks. However, these breaks are not related to apoptotic DNA fragmentation. We propose that p21(CDKN1A) prevents the collapse of replication forks damaged by stabilized topoisomerase I cleavage complexes.

Aphidicolin↗
PubMed2001

Characterization of the stability and folding of H2A.Z chromatin particles: implications for transcriptional activation.

H2A.Z and H2A.1 nucleosome core particles and oligonucleosome arrays were obtained using recombinant versions of these histones and a native histone H2B/H3/H4 complement reconstituted onto appropriate DNA templates. Analysis of the reconstituted nucleosome core particles using native polyacrylamide gel electrophoresis and DNase I footprinting showed that H2A.Z nucleosome core particles were almost structurally indistinguishable from its H2A.1 or native chicken erythrocyte counterparts. While this result is in good agreement with the recently published crystallographic structure of the H2A.Z nucleosome core particle (Suto, R. K., Clarkson, M J., Tremethick, D. J., and Luger, K. (2000) Nat. Struct. Biol. 7, 1121-1124), the ionic strength dependence of the sedimentation coefficient of these particles exhibits a substantial destabilization, which is most likely the result of the histone H2A.Z-H2B dimer binding less tightly to the nucleosome. Analytical ultracentrifuge analysis of the H2A.Z 208-12, a DNA template consisting of 12 tandem repeats of a 208-base pair sequence derived from the sea urchin Lytechinus variegatus 5 S rRNA gene, reconstituted oligonucleosome complexes in the absence of histone H1 shows that their NaCl-dependent folding ability is significantly reduced. These results support the notion that the histone H2A.Z variant may play a chromatin-destabilizing role, which may be important for transcriptional activation.

Chromatin↗
PubMed2001

Recombinational DNA double-strand breaks in mice precede synapsis.

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'.

Animals↗
PubMed2000

Response to RAG-mediated VDJ cleavage by NBS1 and gamma-H2AX.

Genetic disorders affecting cellular responses to DNA damage are characterized by high rates of translocations involving antigen receptor loci and increased susceptibility to lymphoid malignancies. We report that the Nijmegen breakage syndrome protein (NBS1) and histone gamma-H2AX, which associate with irradiation-induced DNA double-strand breaks (DSBs), are also found at sites of VDJ (variable, diversity, joining) recombination-induced DSBs. In developing thymocytes, NBS1 and gamma-H2AX form nuclear foci that colocalize with the T cell receptor alpha locus in response to recombination activating gene (RAG) protein-mediated VDJ cleavage. Our results suggest that surveillance of T cell receptor recombination intermediates by NBS1 and gamma-H2AX may be important for preventing oncogenic translocations.

Amino Acid Sequence↗
PubMed2000

Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139.

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 gamma-H2AX, appears during apoptosis concurrently with the initial appearance of high molecular weight DNA fragments. gamma-H2AX forms before the appearance of internucleosomal DNA fragments and the externalization of phosphatidylserine to the outer membrane leaflet. gamma-H2AX formation is inhibited by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and the inhibitor of caspase-activated DNase, and it is induced when DNase I and restriction enzymes are introduced into cells, suggesting that any apoptotic endonuclease is sufficient to induce gamma-H2AX formation. These results indicate that gamma-H2AX formation is an early chromatin modification following initiation of DNA fragmentation during apoptosis.

Apoptosis↗
PubMed1999

Megabase chromatin domains involved in DNA double-strand breaks in vivo.

The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a specific modified form named gamma-H2AX. An antibody prepared to the unique region of human gamma-H2AX shows that H2AX homologues are phosphorylated not only in irradiated mammalian cells but also in irradiated cells from other species, including Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae. The antibody reveals that gamma-H2AX appears as discrete nuclear foci within 1 min after exposure of cells to ionizing radiation. The numbers of these foci are comparable to the numbers of induced DNA double-strand breaks. When DNA double-strand breaks are introduced into specific partial nuclear volumes of cells by means of a pulsed microbeam laser, gamma-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation, gamma-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that gamma-H2AX forms en masse at chromosomal sites of DNA double-strand breaks. The results further suggest the possible existence of units of higher order chromatin structure involved in monitoring DNA integrity.

Animals↗
PubMed1998

DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.

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 gamma, these components are formed in vivo by several procedures that introduce double-stranded breaks into DNA. gamma-Components, which appeared to be the only major novel components detected by mass or 32PO4 incorporation on acetic acid-urea-Triton X-100-acetic acid-urea-cetyltrimethylammonium bromide or SDS-acetic acid-urea-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. gamma-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 gamma-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 x 10(9) base pairs of a mammalian G1 genome, leads to the gamma-phosphorylation of H2AX distributed over 1% of the chromatin. Thus, about 0.03% of the chromatin appears to be involved per DNA double-stranded break. This value, which corresponds to about 2 x 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, gamma-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.

Amino Acid Sequence↗
PubMed1996

An upstream region of the H2AZ gene promoter modulates promoter activity in different cell types.

Human H2AZ gene promoter fragments that included sequences upstream from the core promoter resulted in decreased activity of reporter constructs transfected into several human cell lines, but increased activity in the undifferentiated human embryonal carcinoma cell line Tera-2. Differentiation of Tera-2 cells in media containing retinoic acid restored the ability of the upstream region to downregulate H2AZ gene promoter activity. Levels of endogenous H2AZ mRNA were also found to be 2.5-fold higher in undifferentiated Tera-2 cells than in differentiated Tera-2 cells. A 128 bp region located 234 to 361 bp upstream from the transcription start site of the H2AZ gene was found to be responsible for the modulation of reporter activity. The upstream region also functioned similarly when removed from the H2AZ gene promoter and inserted upstream of the SV40 promoter in reporter constructs. Gel mobility shift studies of fragments of this region revealed two sequence elements, CTCCTCC and CACGTG, that bound nuclear factors in vitro.

Base Sequence↗
PubMed1995

Characterization of the proximal promoter of the human histone H2A.Z gene.

Histone H2A.Z is a distinct and evolutionarily conserved member of the histone H2A family whose synthesis, in contrast to that of most other histone species, is not dependent on DNA replication. The gene for H2A.Z lacks the signals involved in the 3' processing of replication-linked histone mRNA species and contains introns as well as polyadenylation signals. The H2A.Z gene proximal promoter, a 200-bp region upstream of the transcription start site that provides maximal activity in CAT reporter studies, contains three CCAAT and two GGGCGG elements as well as a consensus TATA element. In vitro DNase I footprint analysis of this region indicated that the central CCAAT and the distal GGGCGG elements were protected by factors present in HeLa nuclear extract. Site-directed mutations of selected promoter elements were generated in the H2A.Z gene promoter region of a CAT reporter construct by a novel one-step PCR procedure. Of the elements examined, the central CCAAT element was found to be the most important determinant of promoter activity; its disruption decreased CAT reporter activity by 65%. Disruption of the proximal CCAAT or the distal GGGCGG elements led to decreases in activity of 40%, while disruption of any of the other examined led to smaller decreases. Gel-mobility shift analysis showed that the three CCAAT elements had overlapping but not identical binding specificities for nuclear factors. The two GGGCGG elements both were found to bind transcription factor Sp1, but the distal element bound Sp1 with higher affinity. The findings show that the central and proximal CCAAT elements and the distal GGGCGG element appear to be the major determinants of the transcriptional activity of the H2A.Z gene.

Base Sequence↗
PubMed1994

Characterization of the human histone H2A.X gene. Comparison of its promoter with other H2A gene promoters.

The human gene for the replication-unlinked histone protein H2A.X is a naturally occurring chimera that contains a replication-unlinked promoter yet produces a stemloop mRNA characteristic of replication-linked histone genes. Consistent with the latter attribute, the H2A.X gene was found to lack introns. The promoter of the H2A.X gene was localized to a 120-base pair region upstream of the transcription start site, a region which included a TATA and two CCAAT sequence elements. The proximal of the two CCAAT elements was shown to be an important determinant of H2A.X gene promoter activity. In a comparative study with the CCAAT elements from the replication-linked H2A.1a gene and the replication-unlinked H2A.Z gene, the proximal CCAAT element of the H2A.X gene was found to bind nuclear factors also bound by CCAAT elements in the latter but not in the former. The specificity of the replication-unlinked H2A.X and H2A.Z gene promoters for CCAAT-binding transcription factors appeared to also reside in short homologous sequences about 10 base pairs away on either side of the CCAAT sequence.

Animals↗
PubMed1994

Expression of a marked H2A histone protein in mammalian cells.

Histone protein sequences are highly conserved. In order to determine whether histones with sequences not found in nature would be tolerated in the chromatin of tissue culture cells, a gene for histone H2A.1a was altered by extending the protein coding region with eight amino acids, including three residues for methionine which are lacking in H2A.1a. Isolated clones of HeLa cells transfected with the gene construct were found to produce a novel protein which was resolved from other histone proteins on AUT-AUC two-dimensional gels. One clone, HeLa-B4, in which the novel protein named H2A.E accounted for about 10% of total H2A protein, was studied further. The linkage of H2A.E mRNA concentrations to the rates of DNA and protein synthesis was found to be the same as that of other replication-linked histone mRNA species. The stability of H2A.E in chromatin as well as the partitioning of nascent H2A.E protein between soluble and nuclear fractions was found to be indistinguishable from that of other histone species. This study shows that histone proteins with sequences other than the conserved sequences found in nature may be utilized in tissue culture cells.

Amino Acid Sequence↗
PubMed1994

Chromosomal localization of the human histone H2A.X gene to 11q23.2-q23.3 by fluorescence in situ hybridization.

The human histone H2A.X gene is unusual in that its transcripts are alternatively processed to yield two species, one a 0.6-kb replication-linked histone mRNA and the other a 1.6-kb polyadenylated mRNA. The H2A.X gene has been localized by fluorescence in situ hybridization to chromosome 11q23.2-q23.3, away from the known clusters of human histone genes on chromosomes 1, 6, and 12. Assignment to chromosome 11 was substantiated by analysis of human-hamster somatic cell hybrid lines. As this work was being completed, an 89-bps sequence overlap was found between the downstream regions of the H2A.X gene and the recently sequenced hydroxymethylbilane (HMB)-synthase gene. The H2A.X and HMB-synthase genes have an unusual arrangement, being transcribed towards each other with their polyadenylation sites 330 bp apart. In addition the HMB-synthase gene contains constitutive and erythroid specific promoters. K562, an erythroid cell line, was found to contain a high concentration of the 1.6-kb polyadenylated H2A.X mRNA.

Animals↗
PubMed1993

Histone H2A.X gene transcription is regulated differently than transcription of other replication-linked histone genes.

Histone H2A.X is a replication-independent histone H2A isoprotein species that is encoded by a transcript alternatively processed at the 3' end to yield two mRNAs: a 0.6-kb mRNA ending with the stem-loop structure characteristic of the mRNAs for replication-linked histone species, and a second, polyadenylated 1.6-kb mRNA ending about 1 kb further downstream (C. Mannironi, W. M. Bonner, and C. L. Hatch, Nucleic Acids Res. 17:9113-9126, 1989). Of the two, the 0.6-kb H2A.X stem-loop mRNA predominates in many cell lines, indicating that the presence of two types of mRNA may not completely account for the replication independence of H2A.X protein synthesis. The ambiguity is resolved by the finding that the level of the 0.6-kb H2A.X mRNA is only weakly downregulated during the inhibition of DNA replication and only weakly upregulated during the inhibition of protein synthesis, while the levels of other replication-linked mRNAs are strongly down- or upregulated under these two conditions. Analysis of the nuclear transcription rates of specific H2A genes showed that while the rates of transcription of replication-linked H2A genes decreased substantially during the inhibition of DNA synthesis and increased substantially during the inhibition of protein synthesis, the rate of H2A.X gene transcription decreased slightly under both conditions. These differences in transcriptional regulation between the H2A.X gene and other replication-linked histone genes are sufficient to account for the differences in regulation of their respective stem-loop mRNAs.

Base Sequence↗
PubMed1990

The human histone H2A.Z gene. Sequence and regulation.

The gene encoding the human basal histone variant H2A.Z has been cloned and sequenced. There is a single functional H2A.Z gene with several pseudogene copies. No other histone genes were found in the 3 kilobases of upstream sequence or in the 0.7 kilobase of downstream sequence. In the upstream region, there are regions of Alu sequences, located about 1375 and 2650 base pairs before the transcription start site. The amount of the H2A.Z transcript is unlinked to DNA replication; however, the amount of the H2A.Z transcript is greatly decreased as proliferating cell cultures become quiescent due in part to a decrease in the rate of transcription. Promoter sequences upstream from the H2A.Z gene have been delineated in IMR-90 cells by chloramphenicol acetyltransferase gene expression. Maximal promoter activity was found in a chloramphenicol acetyltransferase construct that contained 234 base pairs just upstream from the transcription start site. This region includes two GC boxes and three CCAAT boxes as well as a properly positioned TATA box. The organization of the human gene is similar to that of the recently characterized chicken gene (Dalton, S., Robins, A. J., Harvey, R. P., and Wells, J. R. E. (1989) Nucleic Acids Res. 17, 1745-1756). Both have four introns with identical exon-intron borders, but three of the introns in the chicken gene are much longer than those in the human. The promoter regions of the two genes have little overall homology; however, two GC boxes and one of the CCAAT boxes are conserved.

Amino Acid Sequence↗
PubMed1989

H2A.X. a histone isoprotein with a conserved C-terminal sequence, is encoded by a novel mRNA with both DNA replication type and polyA 3' processing signals.

A full length cDNA clone that directs the in vitro synthesis of human histone H2A isoprotein H2A.X has been isolated and sequenced. H2A.X contains 142 amino acid residues, 13 more than human H2A.1. The sequence of the first 120 residues of H2A.X is almost identical to that of human H2A.1. The sequence of the carboxy-terminal 22 residues of H2A.X is unrelated to any known sequence in vertebrate histone H2A; however, it contains a sequence homologous with those of several lower eukaryotes. This homology centers on the carboxy-terminal tetrapeptide which in H2A.X is SerGlnGluTyr. Homologous sequences are found in H2As of three types of yeasts, in Tetrahymena and Drosophila. Seven of the nine carboxy-terminal amino acids of H2A.X are identical with those of S. cerevisiae H2A.1. It is suggested that this H2A carboxy-terminal motif may be present in all eukaryotes. The H2A.X cDNA is 1585 bases long followed by a polyA tail. There are 73 nucleotides in the 5' UTR, 432 in the coding region, and 1080 in the 3' UTR. Even though H2A.X is considered a basal histone, being synthesized in G1 as well as in S-phase, and its mRNA contains polyA addition motifs and a polyA tail, its mRNA also contains the conserved stem-loop and U7 binding sequences involved in the processing and stability of replication type histone mRNAs. Two forms of H2A.X mRNA, consistent with the two sets of processing signals were found in proliferating cell cultures. One, about 1600 bases long, contains polyA; the other, about 575 bases long, lacks polyA. The short form behaves as a replication type histone mRNA, decreasing in amount when cell cultures are incubated with inhibitors of DNA synthesis, while the longer behaves as a basal type histone mRNA.

Amino Acid Sequence↗