Dephosphorylation of histone gamma-H2AX during repair of DNA double-strand breaks in mammalian cells and its inhibition by calyculin A.

The induction of DNA double-strand breaks (DSBs) by ionizing radiation in mammalian chromosomes leads to the phosphorylation of Ser-139 in the replacement histone H2AX, but the molecular mechanism(s) of the elimination of phosphorylated H2AX (called gamma-H2AX) from chromatin in the course of DSB repair remains unknown. We showed earlier that gamma-H2AX cannot be replaced by exchange with free H2AX, suggesting the direct dephosphorylation of H2AX in chromatin by a protein phosphatase. Here we studied the dynamics of dephosphorylation of gamma-H2AX in vivo and found that more than 50% was dephosphorylated in 3 h, but a significant amount of gamma-H2AX could be detected even 6 h after the induction of DSBs. At this time, a significant fraction of the gamma-H2AX nuclear foci co-localized with the foci of RAD50 protein that did not co-localize with replication sites. However, gamma-H2AX could be detected in some cells treated with methyl methanesulfonate which accumulated RAD18 protein at stalled replication sites. We also found that calyculin A inhibited early elimination of gamma-H2AX and DSB rejoining in vivo and that protein phosphatase 1 was able to remove phosphate groups from gamma-H2AX-containing chromatin in vitro. Our results confirm the tight association between DSBs and gamma-H2AX and the coupling of its in situ dephosphorylation to DSB repair.

[Recognition of internal (TTAGGG)n repeats by telomeric protein TRF1 and its role in maintenance of chromosomal stability in Chinese hamster cells]. / Uznavanie vnutrennikh povtorov (TTAGGG)n telomernym belkom TRF1 i ego rol' v podderzhanii stabil'nosti khromosom v kletkakh kitaiskogo khomiachka.

Mammalian telomeres contain long tandem (TTAGGG)n repeats, which are protected by a complex of different proteins. Telomeric repeat-binding factors TRF1 and TRF2 play the key role in protection of telomeres through the formation of terminal loops (called T-loop). A T-loop isolates the 3' strand telomeric end and with this mechanism protects telomeres from the influence of enzymes of DNA reparation and telomere fusions and also interferes with the interaction of telomerase with telomeres. Many vertebrate species also contain large blocks of (TTAGGG)n sequences in pericentric and interstitial chromosome bands. The Chinese hamster genome contains a total of 18 arrays of these non-telomeric internal (TTAGGG)n sequences (ITs). Chromosome bands containing these arrays are unstable and should be protected with the help of another mechanism, rather than that using telomeres. In this study we analysed association of Green Fluorescent Protein (GFP)-tagged TRF1 in Chinese hamster V79 cells with ITs. We found that in these cells GFP-TRF1 associates with ITs in the interphase nucleus. We detected a little overlap between IT-associated GFP-TRF1 and random DSB sites visualized after the treatment of V79 cells with ionizing radiation. We found that the treatment of V79 cells with WM significantly increases the frequency of spontaneous chromosome aberrations. These WM effects are possible due to inhibiting phosphorylation of TRF1 by ATM. TRF1 is known to be eliminated from telomeres by overexpression of TANK1, which induces TRF1 poly(ADP-ribosyl)ation. We transfected V79 cells by plasmid encoding TANK1 and found that the frequency of chromosome rearrangements increased in these cells independently of their treatment by IR. Taken together, our results suggest that TRF1 may be involved in the sequence-specific protection of internal non-telomeric (TTAGGG)n repeats.

Sustained activation of p34(cdc2) is required for noscapine-induced apoptosis.

Mitotic arrest and subsequent apoptosis has been observed in many types of cells treated with anti-microtubule agents. However, the molecular mechanisms underlying the two events as well as their relationship are not well understood; on the contrary, there has been increasing evidence indicating that anti-microtubule agents might induce apoptosis via signaling pathways independent of mitosis. In this study, we found that apoptosis induced by noscapine, an anti-microtubule drug previously shown to cause both mitotic arrest and apoptotic cell death, was blocked by inhibiting p34(cdc2) activity with olomoucine in FM3A murine mammary carcinoma cells or by reducing the level and activity of p34(cdc2) in a mutant cell line FT210 derived from FM3A. Furthermore, transfection of the mutant FT210 cells with wild-type p34(cdc2) restored their ability to undergo mitotic arrest and then apoptosis in response to noscapine. Thus, we conclude that sustained activation of the p34(cdc2) kinase during mitotic arrest is required for subsequent apoptosis induced by noscapine, establishing a link between the two events.

Peptide mass mapping constrained with stable isotope-tagged peptides for identification of protein mixtures.

Through proteolysis and peptide mass determination using mass spectrometry, a peptide mass map (PMM) can be generated for protein identification. However, insufficient peptide mass accuracy and protein sequence coverage limit the potential of the PMM approach for high-throughput, large-scale analysis of proteins. In our novel approach, nonlabile protons in particular amino acid residues were replaced with deuteriums to mass-tag proteins of the S. cerevisiae proteome in a sequence-specific manner. The resulting mass-tagged proteolytic peptides with characteristic mass-split patterns can be identified in the data search using constraints of both amino acid composition and mass-to-charge ratio. More importantly, the mass-tagged peptides can further act as internal calibrants with high confidence in a PMM to identify the parent proteins at modest mass accuracy and low sequence coverage. As a result, the specificity and accuracy of a PMM was greatly enhanced without the need for peptide sequencing or instrumental improvements to obtain increased mass accuracy. The power of PMM has been extended to the unambiguous identification of multiple proteins in a 1D SDS gel band including the identification of a membrane protein.

Visualization of focal nuclear sites of DNA repair synthesis induced by bleomycin in human cells.

In this study, we examined DNA repair synthesis in human cells treated with the radiomimetic drug bleomycin, which efficiently induces double-strand breaks (DSBs). Using tyramide-biotin to amplify fluorescent signals, discrete nuclear foci from the incorporation of 5-iododeoxyuridine (IdU) were detected in proliferating human cells treated with bleomycin. We believe this comes from the repair of DSBs. An increase in the number of foci (>5 per nucleus) was detected in a major fraction (75%) of non-S-phase cells labeled for 30 min with IdU 1 h after the end of bleomycin treatment. The fraction of cells with multiple IdU-containing foci was found to decrease 18 h after treatment. The average number of foci per nucleus detected 1 h after bleomycin treatment was found to decrease twofold between 1 and 3.5 h, indicating that the foci may be associated with the slow component of DSB repair. The presence of DSBs in bleomycin-treated cells was confirmed using antibodies against phosphorylated histone H2AX (gamma-H2AX), which is strictly associated with this type of DNA damage. After treatment with bleomycin, non-S-phase cells also displayed heterogeneous nuclear foci containing tightly bound proliferating cell nuclear antigen (PCNA), suggesting an ongoing process of unscheduled DNA synthesis. PCNA is known to be involved in base excision repair, but a fraction of the PCNA foci may also be associated with DNA synthesis occurring during the repair of DSBs.

Expression of p21Waf1/Cip1 and cyclin D1 is increased in butyrate-resistant HeLa cells.

Sodium butyrate induced cell cycle arrest in mammalian cells through an increase in p21Waf1/Cip1, although another study showed that this arrest is related to pRB signaling. We isolated variants of HeLa cells adapted to growth in 5 mm butyrate. One of these variants, clone 5.1, constitutively expressed elevated levels of p21Waf1/Cip1 when incubated in regular growth medium and in the presence of butyrate. Despite this elevated level of p21Waf1/Cip1, the cells continue to proliferate, albeit at a slower rate than parental HeLa cells. Western blot analyses showed that other cell cycle regulatory proteins were not up-regulated to compensate for the elevated expression of p21Waf1/Cip1. However, cyclin D1 was down-regulated by butyrate in HeLa cells but not in clone 5.1. We conclude that continued expression of cyclin D1 allowed clone 5.1 to grow in the presence of butyrate and elevated levels of p21Waf1/Cip1.

Rapid characterization of DNA oligomers and genotyping of single nucleotide polymorphism using nucleotide-specific mass tags.

Using currently available MS-based methods, accurate mass measurements are essential for the characterization of DNA oligomers. However, there is a lack of specificity in mass peaks when the characterization of individual DNA species in a mass spectrum is dependent solely upon the mass-to-charge ratio (m/z). Here, we utilize nucleotide-specific tagging with stable isotopes to provide internal signatures that quantitatively display the nucleotide content of oligomer peaks in MS spectra. The characteristic mass-split patterns induced by the partially (13)C/(15)N-enriched dNTPs in DNA oligomers indicate the number of labeled precursors and in turn the base substitution in each mass peak, and provide for efficient SNP detection. Signals in mass spectra not only reflect the masses of particular DNA oligomers, but also their specific composition of particular nucleotides. The measurements of mass tags are relative in the mass-split pattern and, hence, the accuracy of the determination of nucleotide substitution is indirectly increased. For high sample throughput, (13)C/(15)N-labeled sequences of interest have been generated, excised in solution and purified for MS analysis in a single-tube format. This method can substantially improve the specificity, accuracy and efficiency of mass spectrometry in the characterization of DNA oligomers and genetic variations.

The cyclin-dependent kinase (CDK) inhibitor flavopiridol inhibits glycogen phosphorylase.

Flavopiridol has been shown to induce cell cycle arrest and apoptosis in various tumor cells in vitro and in vivo. Using immobilized flavopiridol, we identified glycogen phosphorylases (GP) from liver and brain as flavopiridol binding proteins from HeLa cell extract. Purified rabbit muscle GP also bound to the flavopiridol affinity column. GP is the rate-limiting enzyme in intracellular glycogen breakdown. Flavopiridol significantly inhibited the AMP-activated GP-b form of the purified rabbit muscle isoenzyme (IC50 of 1 microM at 0.8 mM AMP), but was less inhibitory to the active phosphorylated form of GP, GP-a (IC50 of 2.5 microM). The AMP-bound GP-a form was poorly inhibited by flavopiridol (40% at 10 microM). Increasing concentrations of the allosteric effector AMP resulted in a linear decrease in the GP-inhibitory activity of flavopiridol suggesting interference between flavopiridol and AMP. In contrast the GP inhibitor caffeine had no effect on the relative GP inhibition by flavopiridol, suggesting an additive effect of caffeine. Flavopiridol also inhibited the phosphorylase kinase-catalyzed phosphorylation of GP-b by inhibiting the kinase in vitro. Flavopiridol thus is able to interfere with both activating modifications of GP-b, AMP activation and phosphorylation. In A549 NSCLC cells flavopiridol treatment caused glycogen accumulation despite of an increase in GP activity, suggesting direct GP inhibition in vivo rather than inhibition of GP activation by phosphorylase kinase. These results suggest that the cyclin-dependent kinase inhibitor flavopiridol interferes with glycogen degradation, which may be responsible for flavopiridol's cytotoxicity and explain its resistance in some cell lines.

A negative regulator of telomere-length protein trf1 is associated with interstitial (TTAGGG)n blocks in immortal Chinese hamster ovary cells.

Telomeres of mammalian chromosomes are composed of long tandem repeats (TTAGGG)n which bind in a sequence-specific manner two proteins-TRF1 and TRF2. In human somatic cells both proteins are mostly associated with telomeres and TRF1 overexpression resulting in telomere shortening. However, chromosomes of some mammalian species, e.g., Chinese hamster, have large interstitial blocks of (TTAGGG)n sequence (IBTs) and the blocks are involved in radiation-induced chromosome instability. In normal somatic cells of these species chromosomes are stable, indicating that the IBTs are protected from unequal homologous recombination. In this study we expressed V5-epitope or green fluorescent protein (GFP)-tagged human TRF1 in different lines of mammalian cells and analyzed distribution of the fusion proteins in interphase nucleus. As expected, transient transfection of human (A549) or African green monkey cells with GFP-N-TRF1 or TRF1-C-V5 plasmids resulted in the appearance in interphase nuclei of multiple faint nuclear dots containing GFP or V5 epitope which we believe to represent telomeres. Transfection of immortalized Chinese hamster ovary (CHO) cell line K1 which have extremely short telomeres with GFP-N-TRF1 plasmid leads to the appearance in interphase nuclei of large GFP bodies corresponding in number to the number of IBTs in these cells. Simultaneous visualization of GFP and IBTs in interphase nuclei of transfected CHO-K1 cells showed colocalization of both signals indicating that expressed TRF1 actually associates with IBTs. These results suggest that TRF1 may serve as general sensor of (TTAGGG)n repeats controlling not only telomeres but also interstitial (TTAGGG)n sequences.

Chromatin structure of telomere domain in human sperm.

Telomeres in human sperm nucleus are clustered at the nuclear periphery. Chromosomes in the sperm are highly condensed with protamines, however, a small portion of DNA remains associated with histones; the role of the nucleohistone is unknown. To examine structure of the telomeric chromatin, the sperm nuclei were treated with micrococcal nuclease. Chromatin released by the digestion was free from protamines, but contained histones and revealed nucleosomal organization. It was enriched with telomeric DNA organized into closely spaced nucleosomes with a periodicity of 148 +/- bp. Thus, while the most of the sperm genome is packed into extremely dense nucleoprotamine structure, at least a part of the telomeric DNA is arranged into nucleosomes and can be released by the nuclease. We suggest that telomeres might be among the first structures in the sperm nucleus that respond to oocyte signals for male pronucleus development at fertilization.

Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification.

Proteolytic peptide mass mapping as measured by mass spectrometry provides a major approach for the identification of proteins. A protein is usually identified by the best match between the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. Without ultrahigh instrumental accuracy, it is possible to increase the specificity of the assignments of particular proteolytic peptides by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence. Here we report this novel method of generating residue-specific mass-tagged proteolytic peptides for accurate and efficient protein identification. Selected amino acids are labeled with 13C/15N/2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tags can then be readily distinguished from other peptides in mass spectra. This method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency, and accuracy for protein identifications.

Identification and characterization of a bovine sperm protein that binds specifically to single-stranded telomeric deoxyribonucleic acid.

Telomere DNA at the physical termini of chromosomes forms a single-stranded 3' overhang. In lower eukaryotes, e.g., ciliated protozoa, this DNA extension is capped by specific proteins that have been structurally and functionally characterized. Much less is known about single-stranded telomere DNA-binding proteins in vertebrates. Here we describe a new protein from bovine sperm designated bsSSTBP that specifically interacts with single-stranded (TTAGGG)(N) DNA. The bsSSTBP was extracted from nuclei by 0.6 M KCl. The native size of this protein, estimated by gel filtration, was 20-40 kDa. SDS-PAGE of the UV cross-linked complex between bsSSTBP and telomere DNA indicated that several polypeptides are involved in complex formation. Bovine sSSTB had high specificity toward nucleotide sequence, since single nucleotide substitutions in the (TTAGGG)(4) substrate suppressed binding. The minimal number of (TTAGGG) repeats required for binding of bsSSTBP was 3, and the protein recognized linear but not folded DNA structures. We propose that the bsSSTBP participates in telomere-telomere interactions and the telomere membrane localization observed in mature sperm. In mammals, somatic telomere-binding proteins are apparently substituted by sperm-specific ones that may lead to a structural reorganization of telomere domains to fulfill functions important during meiosis and fertilization.

Human sperm telomere-binding complex involves histone H2B and secures telomere membrane attachment.

Telomeres are unique chromatin domains located at the ends of eukaryotic chromosomes. Telomere functions in somatic cells involve complexes between telomere proteins and TTAGGG DNA repeats. During the differentiation of germ-line cells, telomeres undergo significant reorganization most likely required for additional specific functions in meiosis and fertilization. A telomere-binding protein complex from human sperm (hSTBP) has been isolated by detergent treatment and was partially purified. hSTBP specifically binds double-stranded telomeric DNA and does not contain known somatic telomere proteins TRF1, TRF2, and Ku. Surprisingly, the essential component of this complex has been identified as a specific variant of histone H2B. Indirect immunofluorescence shows punctate localization of H2B in sperm nuclei, which in part coincides with telomeric DNA localization established by fluorescent in situ hybridization. Anti-H2B antibodies block interactions of hSTBP with telomere DNA, and spH2B forms specific complex with this DNA in vitro, indicating that this protein plays a role in telomere DNA recognition. We propose that hSTBP participates in the membrane attachment of telomeres that may be important for ordered chromosome withdrawal after fertilization.

Stable-isotope-assisted MALDI-TOF mass spectrometry for accurate determination of nucleotide compositions of PCR products.

In parallel with a large-scale sequencing effort, the human genome project will need next-generation tools for accurate and efficient analyses of the enormous pool of DNA sequences. Such analyses are required for (a) validation of DNA sequences, (b) comparison of a parent (known) sequence with a related (unknown) sequence, and (c) characterization of sequence polymorphisms in various genes, especially those associated with genetically inherited human diseases. Here, we report a novel method that combines stable isotope 13C/15N labeling of PCR products of the target sequences with analysis of the mass shifts by mass spectrometry (MS). The mass shift due to the labeling of a single type of nucleotide (i.e., A, T, G, or C) reveals the number of that type of nucleotide in a given DNA fragment. Using this technique, we have accurately determined nucleotide compositions of DNA fragments. The method has also been applied to score a known single-nucleotide polymorphism (SNP). The comparisons of nucleotide compositions determined by our method among homologous sequences are useful in sequence validation, sequence comparison, and characterizations of sequence polymorphisms.

Identification of cytosolic aldehyde dehydrogenase 1 from non-small cell lung carcinomas as a flavopiridol-binding protein.

The synthetic flavone flavopiridol can be cytostatic or cytotoxic to mammalian cells, depending on the concentration of the drug and the duration of exposure. It has been shown to inhibit the cyclin-dependent kinase (CDK) family of cell cycle regulatory enzymes. However, the existence of additional potential targets for drug action remains a matter of interest to define. To identify cellular targets, flavopiridol was immobilized. CDKs, particularly CDK 4, bound weakly to immobilized flavopiridol when ATP was absent but not in its presence. Two proteins with molecular weights of 40 kDa and 120 kDa had high affinities to the immobilized flavopiridol independent of the presence of ATP. They were present in all cell lines analyzed: cervical (HeLa), prostate and non-small cell lung carcinoma (NSCLC) cell lines. A 60-kDa protein, which was present only in NSCLC cells and bound similarly well to immobilized flavopiridol, was identified as cytosolic aldehyde dehydrogenase class 1 (ALDH-1). The level of this protein correlated with the resistance of NSCLC cell lines to cytotoxicity caused by 500 nM flavopiridol but not higher flavopiridol concentrations. Despite binding to ALDH-1, there was no inhibition of dehydrogenase activity by flavopiridol concentrations as high as 20 microM and flavopiridol was not metabolized by ALDH-1. The results suggest that high cellular levels of ALDH-1 may reduce cytotoxicity of flavopiridol and contribute to relative resistance to the drug. This is the first report that flavopiridol binds to proteins other than CDKs.

The high-resolution structure of the triplex formed by the GAA/TTC triplet repeat associated with Friedreich's ataxia.

Expansions of the triplet repeat, GAA/TTC, inside the first intron of the frataxin gene causes Friedreich's ataxia (FRDA). It was of interest to us to examine whether the FRDA repeat forms an unusual DNA structure, since formation of such structure during replication may cause its expansion. Here, we show that the FRDA repeat forms a triplex in which the TTC strand folds on either side of the same GAA strand. We have determined the high-resolution NMR structures of two intramolecularly folded FRDA triplexes, (GAA)2T4(TTC)2T4(CTT)2 and (GAA)2T4(TTC)2T2CT2(CTT)2 with T.A.T and C+.G.C triads. T4 represents a synthetic loop sequence, whereas T2CT2 is the natural loop-folding sequence of the TTC strand. We have also made use of site-specific 15N-labeling of the cytosine residues to investigate their protonation status and their interaction with other protons. We show that the cytosine residues of the Hoogsteen C+.G pairs in this triplex are protonated close to physiological pH. Therefore, it appears that the triplex formation offers a plausible explanation for the expansion of the GAA/TTC repeats in FRDA.

DNA repeats in the human genome.

Repetitive DNA sequences, interspersed throughout the human genome, are capable of forming a wide variety of unusual DNA structures with simple and complex loopfolding patterns. The hairpin formed by the fragile X repeat, (CCG)n, and the bipartite triplex formed by the Friedreich's ataxia repeat, (GAA)n/(TTC)n, show simple loopfolding. On the other hand, the doubly folded hairpin formed by the human centromeric repeat, (AATGG)n, the hairpin G-quartet formed by (TTAGGG)n at the 3' telomere overhang, and the hairpin G-quartet, and hairpin C+.C paired i-motif formed by the insulin minisatellite, [formula: see text] show multiple and complex loopfolding. We have performed high resolution nuclear magnetic resonance (NMR) spectroscopy and in vitro replication to show that unique base-pairing and loopfolding render stability to these unusual structures under physiological conditions. The formation of such stable structures offers a mechanism of unwinding which is advantageous during transcription. For example, the formation of the hairpin G-quartet, and hairpin C+.C paired i-motif upstream of the insulin gene may facilitate transcription. These unusual DNA structures also provide unique 'protein recognition motifs' quite different from a Watson-Crick double helix. For example, the hairpin G-quartet formed by (TTAGGG)n at the 3' telomere overhang is specifically recognized and stabilized by the human repair protein, Ku70/Ku80 hetero-dimer, which may be important in the stability of the telomere. However, the formation of the same unusual DNA structures during replication is likely to cause instability in the lengths of the DNA repeats. If the altered (generally expanded) length enhances the probability of the unusual structure during the next cycle of replication, it further increases the instability of the repeat causing a 'dynamic mutation'. In fact, NMR and in vitro replication studies show that the longer the repeat length the higher is the probability of hairpin formation by the fragile X repeat, (CCG)n. In addition, the hairpin of the fragile X repeat, upstream of the FMR-1 gene, is more susceptible to CpG methylation than its duplex thereby leading to methyl-directed suppression of transcription. Thus, the selective advantage of the unusual structures formed by the DNA repeats in the regulation of gene expression may be offset by the genomic instability caused by the same structures during replication. The repeat number is a critical parameter that helps maintain a balance between the advantage gained from an unusual structure during gene expression and the disadvantage posed by the same structure during replication.

Histone-DNA contacts in structure/function relationships of nucleosomes as revealed by crosslinking.

We describe studies of histone-DNA contacts in the nucleosome using the method of covalent zero length protein-DNA crosslinking. These studies show that in intact nuclei isolated from different sources the linear sequential arrangement of histone-DNA contacts in the nucleosomal core is essentially the same. However, the relative strength of certain contacts varies and correlates with the level of chromatin activity and condensation. These altered contacts are located in the sharply bent regions of the nucleosomal DNA and are supposed to be sensitive to the structural changes that may occur during nucleosome functions. Studies of the mechanism of these alterations revealed that the difference in strength of these contacts is attributed to the different conformational state of the nucleosomal core and is caused by stretching of the nucleosomal DNA upon chromatin decondensation during its activation. Histone-terminal domains may be involved in this process through posttranslational modifications affecting chromatin condensation. The described localization of the histone H2A C-terminal domain in the nucleosome by crosslinking demonstrates the ability of this methodology to determine the location of histone-terminal domains and thereby elucidate their role in nucleosome function. Results of the described experiments suggest that chromatin decondensation may alter the nucleosomal DNA conformation and affect the histone-DNA contacts resulting in a structural transition that may play a role in rendering the nucleosome competent for transcription and/or replication.