scholarly journals Telomere Function and the G-Quadruplex Formation are Regulated by hnRNP U

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 390 ◽  
Author(s):  
Hiroto Izumi ◽  
Keiko Funa
Keyword(s):  
Mammalian Cells ◽  
Binding Protein ◽  
Protein A ◽  
Western Blots ◽  
Telomere Repeat ◽  
G Quadruplex ◽  
Hnrnp U ◽  
Telomere Biology

We examine the role of the heterogenous ribonucleoprotein U (hnRNP U) as a G-quadruplex binding protein in human cell lines. Hypothesizing that hnRNP U is associated with telomeres, we investigate what other telomere-related functions it may have. Telomeric G-quadruplexes have been fully characterized in vitro, but until now no clear evidence of their function or in vivo interactions with proteins has been revealed in mammalian cells. Techniques used were immunoprecipitation, DNA pull-down, binding assay, and Western blots. We identified hnRNP U as a G-quadruplex binding protein. Immunoprecipitations disclosed that endogenous hnRNP U associates with telomeres, and DNA pull-downs showed that the hnRNP U C-terminus specifically binds telomeric G-quadruplexes. We have compared the effect of telomere repeat containing RNA (TERRA) on binding between hnRNP U and telomeric (Tel) or single- stranded Tel (ssTel) oligonucleotides and found that ssTel binds stronger to TERRA than to Tel. We also show that hnRNP U prevents replication protein A (RPA) accumulation at telomeres, and the recognition of telomeric ends by hnRNP suggests that a G-quadruplex promoting protein regulates its accessibility. Thus, hnRNP U-mediated formation has important functions for telomere biology.

scholarly journals Tethering DNA Damage Checkpoint Mediator Proteins Topoisomerase IIβ-binding Protein 1 (TopBP1) and Claspin to DNA Activates Ataxia-Telangiectasia Mutated and RAD3-related (ATR) Phosphorylation of Checkpoint Kinase 1 (Chk1)

2011 ◽  
Vol 286 (22) ◽  
pp. 19229-19236 ◽  
Author(s):  
Laura A. Lindsey-Boltz ◽  
Aziz Sancar
Keyword(s):  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Effector Proteins ◽  
Ataxia Telangiectasia Mutated ◽  
Checkpoint Kinase ◽  
Atr Kinase

The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.

scholarly journals Human STAGA Complex Is a Chromatin-Acetylating Transcription Coactivator That Interacts with Pre-mRNA Splicing and DNA Damage-Binding Factors In Vivo

2001 ◽  
Vol 21 (20) ◽  
pp. 6782-6795 ◽  
Author(s):  
Ernest Martinez ◽  
Vikas B. Palhan ◽  
Agneta Tjernberg ◽  
Elena S. Lymar ◽  
Armin M. Gamper ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Excision Repair ◽  
Binding Protein ◽  
Chromatin Modification ◽  
Hereditary Disease ◽  
Coupled Processes ◽  
Transcription Activators ◽  
Sequence Relationships

ABSTRACT GCN5 is a histone acetyltransferase (HAT) originally identified inSaccharomyces cerevisiae and required for transcription of specific genes within chromatin as part of the SAGA (SPT-ADA-GCN5 acetylase) coactivator complex. Mammalian cells have two distinct GCN5 homologs (PCAF and GCN5L) that have been found in three different SAGA-like complexes (PCAF complex, TFTC [TATA-binding-protein-free TAFII-containing complex], and STAGA [SPT3-TAFII31-GCN5L acetylase]). The composition and roles of these mammalian HAT complexes are still poorly characterized. Here, we present the purification and characterization of the human STAGA complex. We show that STAGA contains homologs of most yeast SAGA components, including two novel human proteins with histone-like folds and sequence relationships to yeast SPT7 and ADA1. Furthermore, we demonstrate that STAGA has acetyl coenzyme A-dependent transcriptional coactivator functions from a chromatin-assembled template in vitro and associates in HeLa cells with spliceosome-associated protein 130 (SAP130) and DDB1, two structurally related proteins. SAP130 is a component of the splicing factor SF3b that associates with U2 snRNP and is recruited to prespliceosomal complexes. DDB1 (p127) is a UV-damaged-DNA-binding protein that is involved, as part of a complex with DDB2 (p48), in nucleotide excision repair and the hereditary disease xeroderma pigmentosum. Our results thus suggest cellular roles of STAGA in chromatin modification, transcription, and transcription-coupled processes through direct physical interactions with sequence-specific transcription activators and with components of the splicing and DNA repair machineries.

scholarly journals Inactivation of the gbpA Gene of Streptococcus mutans Increases Virulence and Promotes In Vivo Accumulation of Recombinations between the Glucosyltransferase B and C Genes

1998 ◽  
Vol 66 (5) ◽  
pp. 2180-2185 ◽  
Author(s):  
Karsten R. O. Hazlett ◽  
Suzanne M. Michalek ◽  
Jeffrey A. Banas
Keyword(s):  
Streptococcus Mutans ◽  
Mutant Strain ◽  
Rat Model ◽  
Binding Protein ◽  
Protein A ◽  
Wild Type ◽  
Mutant Strains

ABSTRACT Glucan-binding protein A (GbpA) of Streptococcus mutanshas been hypothesized to promote sucrose-dependent adherence and the cohesiveness of plaque and therefore to contribute to caries formation. We have analyzed the adherence properties and virulence of isogenicgbpA mutants relative to those of wild-type S. mutans. Contrary to expectations, the gbpA mutant strains displayed enhanced sucrose-dependent adherence in vitro and enhanced cariogenicity in vivo. In vitro, S. mutanswas grown in the presence of [3H]thymidine and sucrose within glass vials. When grown with constant rotation, significantly higher levels of gbpA mutant organisms than of wild type remained adherent to the vial walls. Postgrowth vortexing of rotated cultures significantly decreased adherence of wild-type organisms, whereas the adherence of gbpA mutant organisms was unaffected. In the gnotobiotic rat model, the gbpA mutant strain was hypercariogenic though the colonization levels were not significantly different from those of the wild type. ThegbpA mutant strain became enriched in vivo with organisms that had undergone a recombination involving the gtfB andgtfC genes. The incidence of gtfBC recombinant organisms increased as a function of dietary sucrose availability and was inversely correlated with caries development. We propose that the absence of GbpA elevates the cariogenic potential of S. mutans by altering the structure of plaque. However, the hypercariogenic plaque generated by gbpA mutant organisms may be suboptimal for S. mutans, leading to the accumulation of gtfBC recombinants whose reduced glucosyltransferase activity restores a less cariogenic plaque structure.

scholarly journals Telomere DNA G-quadruplex folding within actively extending human telomerase

2019 ◽  
Vol 116 (19) ◽  
pp. 9350-9359 ◽  
Author(s):  
Linnea I. Jansson ◽  
Jendrik Hentschel ◽  
Joseph W. Parks ◽  
Terren R. Chang ◽  
Cheng Lu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Time Series Data ◽  
Resonance Energy ◽  
Series Data ◽  
Dna Repeats ◽  
Telomere Repeat ◽  
G Quadruplex ◽  
Dna Primers

Telomerase reverse transcribes short guanine (G)-rich DNA repeat sequences from its internal RNA template to maintain telomere length. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays, we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using GQ-stabilizing K+ versus GQ-destabilizing Li+ salts yielded changes in DNA product profiles consistent with formation of GQ structures within the telomerase–DNA complex. Addition of the telomerase processivity factor POT1–TPP1 altered the DNA product profile, but was not sufficient to recover full activity in the presence of Li+ cations. This result suggests GQ folding synergizes with POT1–TPP1 to support telomerase function. Single-molecule Förster resonance energy transfer experiments reveal complex DNA structural dynamics during real-time catalysis in the presence of K+ but not Li+, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we globally fit telomerase time-series data to a kinetic model that converges to a set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis, and provide a detailed characterization of GQ modulation of polymerase function.

scholarly journals CREB-Binding Protein Acetylates Hematopoietic Transcription Factor GATA-1 at Functionally Important Sites

1999 ◽  
Vol 19 (5) ◽  
pp. 3496-3505 ◽  
Author(s):  
Hsiao-Ling Hung ◽  
Jason Lau ◽  
Alexander Y. Kim ◽  
Mitchell J. Weiss ◽  
Gerd A. Blobel
Keyword(s):  
Transcription Factor ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Binding Protein ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Creb Binding Protein ◽  
Erythroid Cell Differentiation

ABSTRACT The transcription factor GATA-1 is a key regulator of erythroid-cell differentiation and survival. We have previously shown that the transcriptional cofactor CREB-binding protein (CBP) binds to the zinc finger domain of GATA-1, markedly stimulates the transcriptional activity of GATA-1, and is required for erythroid differentiation. Here we report that CBP, but not p/CAF, acetylates GATA-1 at two highly conserved lysine-rich motifs present at the C-terminal tails of both zinc fingers. Using [3H]acetate labelling experiments and anti-acetyl lysine immunoprecipitations, we show that GATA-1 is acetylated in vivo at the same sites acetylated by CBP in vitro. In addition, we show that CBP stimulates GATA-1 acetylation in vivo in an E1A-sensitive manner, thus establishing a correlation between acetylation and transcriptional activity of GATA-1. Acetylation in vitro did not alter the ability of GATA-1 to bind DNA, and mutations in either motif did not affect DNA binding of GATA-1 expressed in mammalian cells. Since certain functions of GATA-1 are revealed only in an erythroid environment, GATA-1 constructs were examined for their ability to trigger terminal differentiation when introduced into a GATA-1-deficient erythroid cell line. We found that mutations in either acetylation motif partially impaired the ability of GATA-1 to induce differentiation while mutations in both motifs abrogated it completely. Taken together, these data indicate that CBP is an important cofactor for GATA-1 and suggest a novel mechanism in which acetylation by CBP regulates GATA-1 activity in erythroid cells.

scholarly journals A Novel G-Quadruplex Binding Protein in Yeast—Slx9

Molecules ◽  
2019 ◽  
Vol 24 (9) ◽  
pp. 1774 ◽  
Author(s):  
Silvia Götz ◽  
Satyaprakash Pandey ◽  
Sabrina Bartsch ◽  
Stefan Juranek ◽  
Katrin Paeschke
Keyword(s):  
Binding Protein ◽  
Sequence Motifs ◽  
Dna And Rna ◽  
G4 Dna ◽  
G Quadruplex ◽  
High Thermodynamic Stability ◽  
Guanine Base ◽  
Regulatory Functions

G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.

scholarly journals Transferrin-binding protein B isolated from Neisseria meningitidis discriminates between apo and diferric human transferrin

1998 ◽  
Vol 334 (1) ◽  
pp. 269-273 ◽  
Author(s):  
Ian C. BOULTON ◽  
Andrew R. GORRINGE ◽  
Nigel ALLISON ◽  
Andrew ROBINSON ◽  
Beatrice GORINSKY ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Molecular Mass ◽  
Binding Protein ◽  
Protein A ◽  
Gel Filtration ◽  
Free Form ◽  
Stable Complex ◽  
Receptor Complex

Neisseria meningitidis utilization of human serum transferrin (hTF)-bound iron is an important pathogenicity determinant. The efficiency of this system would clearly be increased through preferential binding of diferric hTF over the iron-free form. To characterize this process, functionally active meningococcal transferrin-binding protein A (TbpA) and TbpB have been purified from N. meningitidis using a novel purification procedure. The association of isolated Tbps and Tbps in the presence of hTF was investigated by gel filtration. Co-purified TbpA+B formed a complex of molecular mass 300 kDa which bound 1–2 molecules of hTF. Purified TbpA formed a complex of 200 kDa, indicating association as a dimer, whereas TbpB aggregated to form multimers of variable sizes. On recombining TbpA and TbpB, a stable complex of equivalent size to co-purified TbpA+B was formed. This complex may be composed of a single TbpA dimer and 1 molecule of TbpB. The technique of surface plasmon resonance (SPR) was used to demonstrate clearly that TbpB of either high (85 kDa) or low (68 kDa) molecular-mass preferentially bound diferric hTF in comparison with iron-free hTF. This selectivity was not observed with TbpA, but was found at low levels with co-purified TbpA+B. Individual TbpA and TbpB, recombined in a 1:1 molecular ratio, showed iron-mediated discriminatory binding at an intermediate level. SPR was also used to show that TbpA and TbpB bound to distinct regions of hTF, and that prior saturation with TbpB reduced subsequent TbpA binding. The results demonstrated that hTF bound more TbpA than TbpB, with an approximate ratio of 2:1. We have demonstrated that in vitro, TbpA+B exists as a receptor complex composed of a TbpA dimer and one molecule of TbpB, and that TbpB selectively binds diferric hTF. We propose that, in vivo, TbpA and TbpB also exist as a receptor complex, with TbpB selectively binding diferric hTF, bringing it close to TbpA, the transmembrane component, where the ferric iron can be transported to the periplasm.

Translocation of a UV-damaged DNA binding protein into a tight association with chromatin after treatment of mammalian cells with UV light

1997 ◽  
Vol 110 (10) ◽  
pp. 1159-1168 ◽  
Author(s):  
V.R. Otrin ◽  
M. McLenigan ◽  
M. Takao ◽  
A.S. Levine ◽  
M. Protic
Keyword(s):  
Dna Binding ◽  
Genomic Dna ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Uv Treatment ◽  
Tight Association ◽  
Damaged Dna

A UV-damaged DNA binding protein (UV-DDB) is the major source of UV-damaged DNA binding activity in mammalian cell extracts. This activity is defective in at least some xeroderma pigmentosum group E (XP-E) patients; microinjection of the UV-DDB protein into their fibroblasts corrects nucleotide excision repair (NER). In an in vitro reconstituted NER system, small amounts of UV-DDB stimulate repair synthesis a few fold. After exposure to UV, mammalian cells show an early dose-dependent inhibition of the extractable UV-DDB activity; this inhibition may reflect a tight association of the binding protein with UV-damaged genomic DNA. To investigate the dynamics and location of UV-DDB with respect to damaged chromatin in vivo, we utilized nuclear fractionation and specific antibodies and detected translocation of the p127 component of UV-DDB from a loose to a tight association with chromatinized DNA immediately after UV treatment. A similar redistribution was found for other NER proteins, i.e. XPA, RP-A and PCNA, suggesting their tighter association with genomic DNA after UV. These studies revealed a specific protein-protein interaction between UV-DDB/p127 and RP-A that appears to enhance binding of both proteins to UV-damaged DNA in vitro, providing evidence for the involvement of UV-DDB in the damage-recognition step of NER. Moreover, the kinetics of the reappearance of extractable UV-DDB activity after UV treatment of human cells with differing repair capacities positively correlate with the cell's capacity to repair 6–4 pyrimidine dimers (6–4 PD) in the whole genome, a result consistent with an in vivo role for UV-DDB in recognizing this type of UV lesion.

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