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PubMed1997

Control of telomere length by the human telomeric protein TRF1.

Human telomeres, the nucleoprotein complexes at chromosome ends, consist of tandem arrays of TTAGGG repeats bound to specific proteins. In normal human cells, telomeres shorten with successive cell divisions, probably due to the terminal sequence loss that accompanies DNA replication. In tumours and immortalized cells, this decline is halted through the activation of telomerase, a reverse transcriptase that extends the telomeric TTAGGG-repeat arrays. Telomere length is stable in several immortal human-cell lines, suggesting that a regulatory mechanism exists for limiting telomere elongation by telomerase. Here we show that the human telomeric-repeat binding factor TRF1 (ref. 8) is involved in this regulation. Long-term overexpression of TRF1 in the telomerase-positive tumour-cell line HT1080 resulted in a gradual and progressive telomere shortening. Conversely, telomere elongation was induced by expression of a dominant-negative TRF1 mutant that inhibited binding of endogenous TRF1 to telomeres. Our results identify TRF1 as a suppressor of telomere elongation and indicate that TRF1 is involved in the negative feedback mechanism that stabilizes telomere length. As TRF1 does not detectably affect the expression of telomerase, we propose that the binding of TRF1 controls telomere length in cis by inhibiting the action of telomerase at the ends of individual telomeres.

DNA-Binding Proteins↗

PubMed2000

Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation.

Telomeres are specialized nucleoprotein complexes that serve as protective caps of linear eukaryotic chromosomes. Loss of telomere function is associated with rampant genetic instability and loss of cellular viability and renewal potential. The telomere also participates in processes of chromosomal repair, as evidenced by the 'capture' or de novo synthesis of telomere repeats at double-stranded breaks and by the capacity of yeast telomeres to serve as repositories of essential components of the DNA repair machinery, particularly those involved in non-homologous end-joining (NHEJ). Here we used the telomerase-deficient mouse, null for the essential telomerase RNA gene (Terc), to assess the role of telomerase and telomere function on the cellular and organismal response to ionizing radiation. Although the loss of telomerase activity per se had no discernable impact on the response to ionizing radiation, the emergence of telomere dysfunction in late-generation Terc-/- mice imparted a radiosensitivity syndrome associated with accelerated mortality. On the cellular level, the gastrointestinal crypt stem cells and primary thymocytes showed increased rates of apoptosis, and mouse embryonic fibroblasts (MEFs) showed diminished dose-dependent clonogenic survival. The radiosensitivity of telomere dysfunctional cells correlated with delayed DNA break repair kinetics, persistent chromosomal breaks and cytogenetic profiles characterized by complex chromosomal aberrations and massive fragmentation. Our findings establish a intimate relationship between functionally intact telomeres and the genomic, cellular and organismal response to ionizing radiation.

Animals↗

PubMed1997

Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2.

Human telomeres are composed of long arrays of TTAGGG repeats that form a nucleoprotein complex required for the protection and replication of chromosome ends. One component of human telomeres is the TTAGGG repeat binding factor 1 (TRF1), a ubiquitously expressed protein, related to the protooncogene Myb, that is present at telomeres throughout the cell cycle. Recent evidence has implicated TRF1 in the control of telomere length. TRF1 is proposed to be an inhibitor of telomerase, acting in cis to limit the elongation of individual chromosome ends. Here we report the cloning of TRF2, a distant homologue of TRF1 that carries a very similar Myb-related DNA-binding motif. Like TRF1, TRF2 was ubiquitously expressed, bound specifically to duplex TTAGGG repeats in vitro and localized to all human telomeres in metaphase chromosomes. TRF2 was shown to have an architecture similar to that of TRF1 in that it carries a C-terminal Myb motif and a large TRF1-related dimerization domain near its N terminus. However, the dimerization domains of TRF1 and TRF2 did not interact, suggesting that these proteins exist predominantly as homodimers. While having similar telomere binding activity and domain organization, TRF2 differed from TRF1 in that its N terminus was basic rather than acidic, and TRF2 was much more conserved than TRF1. The results indicate that the TTAGGG repeat arrays at the ends of human and mouse chromosomes bind to two related proteins. Because TRF1 and TRF2 showed significant differences, we suggest that these factors have distinct functions at telomeres.

Amino Acid Sequence↗

PubMed2003

The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif.

The human protein apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3G (APOBEC3G), also known as CEM-15, mediates a newly described form of innate resistance to retroviral infection by catalyzing the deamination of deoxycytidine to deoxyuridine in viral cDNA replication intermediates. Because DNA deamination takes place after virus entry into target cells, APOBEC3G function is dependent on its association with the viral nucleoprotein complexes that synthesize cDNA and must therefore be incorporated into virions as they assemble in infected cells. Here we show that the HIV-1 virion infectivity factor (Vif) protein protects the virus from APOBEC3G-mediated inactivation by preventing its incorporation into progeny virions, thus allowing the ensuing infection to proceed without DNA deamination. In addition to helping exclude APOBEC3G from nascent virions, Vif also removes APOBEC3G from virus-producing cells by inducing its ubiquitination and subsequent degradation by the proteasome. Our findings indicate that pharmacologic strategies aimed at stabilizing APOBEC3G in HIV-1 infected cells should be explored as potential HIV/AIDS therapeutics.

APOBEC-3G Deaminase↗

PubMed2005

Exploring icosahedral virus structures with VIPER.

Virus structures are megadalton nucleoprotein complexes with an exceptional variety of protein-protein and protein-nucleic-acid interactions. Three-dimensional crystal structures of over 70 virus capsids, from more than 20 families and 30 different genera of viruses, have been solved to near-atomic resolution. The enormous amount of information contained in these structures is difficult to access, even for scientists trained in structural biology. Virus Particle Explorer (VIPER) is a web-based catalogue of structural information that describes the icosahedral virus particles. In addition to high-resolution crystal structures, VIPER has expanded to include virus structures obtained by cryo-electron microscopy (EM) techniques. The VIPER database is a powerful resource for virologists, microbiologists, virus crystallographers and EM researchers. This review describes how to use VIPER, using several examples to show the power of this resource for research and educational purposes.

Capsid↗

PubMed2006

Reconstitution of chromatin transcription with purified components reveals a chromatin-specific repressive activity of p300.

Here we describe an in vitro chromatin transcription system in which chromatin assembly and transcription are carried out with purified and defined factors. With basal (also known as general) transcription factors and sequence-specific DNA-binding activators, we observed chromatin-specific, activation domain-dependent transcription. We then examined the biochemical function of purified p300 in the absence of the endogenous factor and other related activities and found, unexpectedly, that p300 has a chromatin-specific, transcriptional repression activity that can be relieved by the addition of acetyl-CoA. This p300-mediated repression is reversible, requires the p300 bromodomain but not the acetyltransferase region, and does not involve the formation of a stable, nuclease-resistant nucleoprotein complex. Hence, the mechanism of transcriptional repression by p300 is distinct from that of histone H1, PARP-1 or Sir2. These findings reveal a novel chromatin-specific repressive function of p300.

Acetyl Coenzyme A↗

PubMed2002

A human interstitial telomere associates in vivo with specific TRF2 and TIN2 proteins.

Mammalian telomeres are composed of long arrays of TTAGGG repeats that form a nucleoprotein complex which protects the chromosome ends. Human telomere function is known to require two TTAGGG repeat factors, TRF1 and TRF2, and several interacting proteins, but the mechanism by which the DNA/protein complex prevents end to end fusion in vivo has not been elucidated. In order to better understand the role of specific telomere-associated proteins in the organisation of chromosome ends, we have studied a patient with a rare chromosome rearrangement that has given rise to an interstitial telomere. Using specific antibodies and immuno-FISH on unfixed metaphase chromosomes, we show that the proteins TRF2 and TIN2 (TIN2 interacts with TRF1) co-localise with the interstitial TTAGGG repeats. Our results demonstrate, for the first time in humans, that TRF2 and TIN2 proteins associate with interstitial duplex TTAGGG repeats, in vivo. They confirm that double stranded-telomeric repeats, even when complexed with specific proteins, are not sufficient to create a functional telomere. Finally, they suggest a possible role for proteins in stabilising interstitial TTAGGG repeats.

Chromosome Aberrations↗

PubMed2004

Positive and negative regulation of SMC-DNA interactions by ATP and accessory proteins.

Structural maintenance of chromosomes (SMC) proteins are central regulators of higher-order chromosome dynamics from bacteria to humans. The Bacillus subtilis SMC (BsSMC) homodimer adopts a V-shaped structure with an ATP-binding catalytic domain at each end. We report here that two small proteins, ScpA and ScpB, associate with the catalytic domains of BsSMC in an ordered fashion and suppress its ATPase activity. When combined with a 'transition state' mutant of BsSMC that poorly hydrolyzes ATP, ScpA promotes stable engagement of two catalytic domains in an ATP-dependent manner. In solution, this occurs intramolecularly and closes the DNA-entry gate of an SMC dimer. ScpB further stabilizes this conformation and prevents BsSMC from binding to double-stranded DNA (dsDNA). In contrast, when the mutant BsSMC is first allowed to interact with dsDNA, subsequent addition of ScpA leads to assembly of large nucleoprotein complexes, possibly by stabilizing intermolecular engagement of the catalytic domains from different SMC dimers. We propose that the ATP-modulated engagement/disengagement cycle of SMC proteins plays both positive and negative roles in their dynamic interactions with dsDNA.

Adenosine Triphosphatases↗

PubMed2004

The carboxy-terminal portion of TnsC activates the Tn7 transposase through a specific interaction with TnsA.

Tn7 transposition requires the assembly of a nucleoprotein complex containing four self-encoded proteins, transposon ends, and target DNA. Within this complex, TnsC, the molecular switch that regulates transposition, and TnsA, one part of the transposase, interact directly. Here, we demonstrate that residues 504-555 of TnsC are responsible for TnsA/TnsC interaction. The crystal structure of the TnsA/TnsC(504-555) complex, resolved to 1.85 A, illustrates the burial of a large hydrophobic patch on the surface of TnsA. One consequence of sequestering this patch is a marked increase in the thermal stability of TnsA as shown by differential scanning calorimetry. A model based on the complex structure suggested that TnsA and a slightly longer version of the cocrystallized TnsC fragment (residues 495-555) might cooperate to bind DNA, a prediction confirmed using gel mobility shift assays. Donor DNA binding by the TnsA/TnsC(495-555) complex is correlated with the activation of the TnsAB transposase, as measured by double-stranded DNA cleavage assays, demonstrating the importance of the TnsA/TnsC interaction in affecting Tn7 transposition.

Amino Acid Sequence↗

PubMed2005

Impairment of replication fork progression mediates RNA polII transcription-associated recombination.

Homologous recombination safeguards genome integrity, but it can also cause genome instability of important consequences for cell proliferation and organism development. Transcription induces recombination, as shown in prokaryotes and eukaryotes for both spontaneous and developmentally regulated events such as those responsible for immunoglobulin class switching. Deciphering the molecular basis of transcription-associated recombination (TAR) is important in understanding genome instability. Using novel plasmid-borne recombination constructs in Saccharomyces cerevisiae, we show that RNA polymerase II (RNAPII) transcription induces recombination by impairing replication fork progression. RNAPII transcription concomitant to head-on oncoming replication causes a replication fork pause (RFP) that is linked to a significant increase in recombination. However, transcription that is codirectional with replication has little effect on replication fork progression and recombination. Transcription occurring in the absence of replication does not affect either recombination or replication fork progression. The Rrm3 helicase, which is required for replication fork progression through nucleoprotein complexes, facilitates replication through the transcription-dependent RFP site and reduces recombination. Therefore, our work provides evidence that one mechanism responsible for TAR is RNAP-mediated replication impairment.

DNA Helicases↗

PubMed2003

Priming the nucleosome: a role for HMGB proteins?

The high-mobility-group B (HMGB) chromosomal proteins are characterized by the HMG box, a DNA-binding domain that both introduces a tight bend into DNA and binds preferentially to a variety of distorted DNA structures. The HMGB proteins seem to act primarily as architectural facilitators in the manipulation of nucleoprotein complexes; for example, in the assembly of complexes involved in recombination and transcription. Recent genetic and biochemical evidence suggests that these proteins can facilitate nucleosome remodelling. One mechanism by which HMGB proteins could prime the nucleosome for migration is to loosen the wrapped DNA and so enhance accessibility to chromatin-remodelling complexes and possibly also to transcription factors. By constraining a tight loop of untwisted DNA at the edge of a nucleosome, an HMGB protein could induce movements in the contacts between certain core histones that would result in an overall change in nucleosome structure.

HMGB Proteins↗

PubMed2001

Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory specificity.

Fos and Jun family proteins regulate the expression of a myriad of genes in a variety of tissues and cell types. This functional versatility emerges from their interactions with related bZIP proteins and with structurally unrelated transcription factors. These interactions at composite regulatory elements produce nucleoprotein complexes with high sequence-specificity and regulatory selectivity. Several general principles including binding cooperativity and conformational adaptability have emerged from studies of regulatory complexes containing Fos-Jun family proteins. The structural properties of Fos-Jun family proteins including opposite orientations of heterodimer binding and the ability to bend DNA can contribute to the assembly and functions of such complexes. The cooperative recruitment of transcription factors, coactivators and chromatin remodeling factors to promoter and enhancer regions generates multiprotein transcription regulatory complexes with cell- and stimulus-specific transcriptional activities. The gene-specific architecture of these complexes can mediate the selective control of transcriptional activity.

Animals↗

PubMed2004

Mycobacterium tuberculosis DnaA initiator protein: purification and DNA-binding requirements.

The Mycobacterium tuberculosis oriC (the origin of chromosomal replication) region contains 13 non-perfect DnaA boxes. The M. tuberculosis initiator protein, DnaA, was overexpressed in Escherichia coli as a soluble His-tagged fusion protein. The purified protein His6MtDnaA was investigated for its binding properties to DnaA boxes from the oriC region. Gel retardation demonstrated that the DnaA from M. tuberculosis requires two DnaA boxes for efficient binding. Electron microscopy as well as DNase I footprinting showed that the His6MtDnaA protein binds to four specific regions, which correspond to the location of 11 out of 13 previously identified DnaA boxes within the M. tuberculosis oriC. Probably, in M. tuberculosis, DnaA molecules by co-operative binding of numerous 'non-perfect' DnaA boxes assemble along the oriC region and subsequently form a massive nucleoprotein complex.

Bacterial Proteins↗

PubMed2005

Highly conserved proximal promoter element harbouring paired Sox9-binding sites contributes to the tissue- and developmental stage-specific activity of the matrilin-1 gene.

The matrilin-1 gene has the unique feature that it is expressed in chondrocytes in a developmental stage-specific manner. Previously, we found that the chicken matrilin-1 long promoter with or without the intronic enhancer and the short promoter with the intronic enhancer restricted the transgene expression to the columnar proliferative chondroblasts and prehypertrophic chondrocytes of growth-plate cartilage in transgenic mice. To study whether the short promoter shared by these transgenes harbours cartilage-specific control elements, we generated transgenic mice expressing the LacZ reporter gene under the control of the matrilin-1 promoter between -338 and +67. Histological analysis of the founder embryos demonstrated relatively weak transgene activity in the developing chondrocranium, axial and appendicular skeleton with highest level of expression in the columnar proliferating chondroblasts and prehypertrophic chondrocytes. Computer analysis of the matrilin-1 genes of amniotes revealed a highly conserved Pe1 (proximal promoter element 1) and two less-conserved sequence blocks in the distal promoter region. The inverted Sox motifs of the Pe1 element interacted with chondrogenic transcription factors Sox9, L-Sox5 and Sox6 in vitro and another factor bound to the spacer region. Point mutations in the Sox motifs or in the spacer region interfered with or altered the formation of nucleoprotein complexes in vitro and significantly decreased the reporter gene activity in transient expression assays in chondrocytes. In vivo occupancy of the Sox motifs in genomic footprinting in the expressing cell type, but not in fibroblasts, also supported the involvement of Pe1 in the tissue-specific regulation of the gene. Our results indicate that interaction of Pe1 with distal DNA elements is required for the high level, cartilage- and developmental stage-specific transgene expression.

Animals↗

PubMed1996

Two regulatory elements of similar structure and placed in tandem account for the repressive activity of the first intron of the human apolipoprotein A-II gene.

Recent reports indicate that apolipoprotein (apo) A-II, the second most abundant protein of high-density lipoproteins, plays a crucial role in counteracting the beneficial effect of apo A-I against atherogenesis. Transcription of the human apo A-II gene is controlled by an enhancer comprising 14 regulatory elements located upstream of its promoter whereas the first intron of this gene behaves as a silencer. Here we show that two sequence elements account for the repressive activity of this intron and correspond to negative regulatory elements termed NRE I and NRE II. The activity of intron I and the nuclear proteins binding to NRE I and II are encountered in hepatic cells but not in non-hepatic cells studied here. Both NREs form nucleoprotein complexes of very similar physicochemical characteristics and bind the same or closely related proteins. Site-directed mutagenesis, transient transfection and gel-shift analysis experiments indicate that both NREs exhibit similar structures, being composed of two sites required for maximal activity and optimal binding of transcription factors. Therefore two negative regulatory elements of similar structure and function, placed in tandem, account for the repressive activity of the first intron of the human apo A-II gene. These NREs do not exhibit structural similarity with known NREs of other genes.

Animals↗

PubMed1997

Reconstitution of native-like nucleosome core particles from reversed-phase-HPLC-fractionated histones.

We have reconstituted nucleosome core particles from reversed-phase-HPLC-purified chicken erythrocyte core histones and 145 bp random-sequence DNA fragments. Characterization of the resulting nucleoprotein complexes by sedimentation velocity, CD and DNase I footprinting showed that they are structurally indistinguishable from native nucleosome core particles. Furthermore, we have shown that the ability to reproduce these native-like structural features in these reconstituted nucleosome core particles is basically independent of the biological source or the method used (i.e. salt versus acid) for the extraction of histones before their HPLC fractionation. The usefulness and relevance of this approach for the reconstitution of native-like chromatin structures from histone types (histone variants/post-translationally modified histones), which are usually available only in relatively small amounts, is discussed.

Animals↗

PubMed2001

HMG1 and 2: architectural DNA-binding proteins.

HMG1 and 2 (high mobility group proteins 1 and 2; renamed HMGB1 and 2) contain two DNA-binding HMG-box domains (A and B) and a long acidic C-terminal domain. They bind DNA without sequence specificity, but have a high affinity for bent or distorted DNA, and bend linear DNA. The individual A and B boxes (which, although broadly similar, show both structural and functional differences) exhibit many of the structure-specific properties of the whole protein. The acidic tail modulates the affinity of the tandem HMG boxes in HMG1 and 2 for a variety of DNA targets, including four-way junctions, but not distorted DNA minicircles, to which the proteins bind with very high affinity. HMG1 and 2 appear to play important architectural roles in the assembly of nucleoprotein complexes in a variety of biological processes, for example V(D)J recombination, the initiation of transcription, and DNA repair.

Binding Sites↗

PubMed2005

Multifaceted role of the Saccharomyces cerevisiae Srs2 helicase in homologous recombination regulation.

Homologous recombination (HR) is a major pathway for the elimination of DNA DSBs (double-strand breaks) induced by high-energy radiation and chemicals, or that arise due to endogenous damage and stalled DNA replication forks. If not processed properly, DSBs can lead to cell death, chromosome aberrations and tumorigenesis. Even though HR is important for genome maintenance, it can also interfere with other DNA repair mechanisms and cause gross chromosome rearrangements. In addition, HR can generate DNA or nucleoprotein intermediates that elicit prolonged cell-cycle arrest and sometimes cell death. Genetic analyses in the yeast Saccharomyces cerevisiae have revealed a central role of the Srs2 helicase in preventing untimely HR events and in inhibiting the formation of potentially deleterious DNA structures or nucleoprotein complexes upon DNA replication stress. Paradoxically, efficient repair of DNA DSBs by HR is dependent on Srs2. In this paper, we review recent molecular studies aimed at deciphering the multifaceted role of Srs2 in HR and other cellular processes. These studies have provided critical insights into how HR is regulated in order to preserve genomic integrity and promote cell survival.

Cytoskeleton↗