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1.
2.
Karamysheva ZN Surovtseva YV Vespa L Shakirov EV Shippen DE 《The Journal of biological chemistry》2004,279(46):47799-47807
Little is known about the protein composition of plant telomeres. We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins hTRF1 and hTRF2. hTRF1/hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro. Twelve Arabidopsis genes fitting this criterion, dubbed TRF-like (TRFL), fell into two distinct gene families. Notably, TRFL family 1 possessed a highly conserved region C-terminal to the Myb domain called Myb-extension (Myb-ext) that is absent in TRFL family 2 and hTRF1/hTRF2. Immunoprecipitation experiments revealed that recombinant proteins from TRFL family 1, but not those from family 2, formed homodimers and heterodimers in vitro. DNA binding studies with isolated C-terminal fragments from TRFL family 1 proteins, but not family 2, showed specific binding to double-stranded plant telomeric DNA in vitro. Removal of the Myb-ext domain from TRFL1, a family 1 member, abolished DNA binding. However, when the Myb-ext domain was introduced into the corresponding region in TRFL3, a family 2 member, telomeric DNA binding was observed. Thus, Myb-ext is required for binding plant telomeric DNA and defines a novel class of proteins in Arabidopsis. 相似文献
3.
The TRF2-Rap1 complex suppresses non-homologous end joining and interacts with DNAPK-C to prevent end joining. We previously demonstrated that hTRF2 is a double strand telomere binding protein that forms t-loops in vitro and recognizes three- and four-way junctions independent of DNA sequence. How the DNA binding characteristics of hTRF2 to DNA is altered in the presence of hRap1 however is not known. Here we utilized EM and quantitative gel retardation to characterize the DNA binding properties of hRap1 and the TRF2-Rap1 complex. Both gel filtration chromatography and mass analysis from two-dimensional projections showed that the TRF2-Rap1 complex exists in solution and binds to DNA as a complex consisting of four monomers each of hRap1 and hTRF2. EM revealed for the first time that hRap1 binds to DNA templates in the absence of hTRF2 with a preference for double strand-single strand junctions in a sequence independent manner. When hTRF2 and hRap1 are in a complex, its affinity for ds telomeric sequences is 2-fold higher than TRF2 alone and more than 10-fold higher for telomeric 3′ ends. This suggests that as hTRF2 recruits hRap1 to telomeric sequences, hRap1 alters the affinity of hTRF2 and its binding preference on telomeric DNA. Moreover, the TRF2-Rap1 complex has higher ability to re-model telomeric DNA than either component alone. This finding underlies the importance of complex formation between hRap1 and hTRF2 for telomere function and end protection. 相似文献
4.
Rescue of an hTERT mutant defective in telomere elongation by fusion with hPot1 总被引:9,自引:0,他引:9
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Armbruster BN Linardic CM Veldman T Bansal NP Downie DL Counter CM 《Molecular and cellular biology》2004,24(8):3552-3561
The protein hPot1 shares homology with telomere-binding proteins in lower eukaryotes and associates with single-stranded telomeric DNA in vitro as well as colocalizing with telomere-binding proteins in vivo. We now show that hPot1 is coimmunoprecipitated with telomeric DNA and that stable expression of this protein in telomerase-positive cells results in telomere elongation, supporting the idea that hPot1 is a bona fide mammalian telomere-binding protein. We previously found that mutations in the N-terminal DAT domain of the hTERT catalytic subunit of telomerase rendered the enzyme catalytically active but unable to elongate telomeres in vivo. This phenotype could be partially rescued by fusion with the double-stranded telomeric protein hTRF2. Given that hPot1 binds to single-stranded DNA in vitro (at the same site that hTERT binds to in vivo), we addressed whether fusion of hPot1 can rescue the DAT mutations more efficiently than that of hTRF2. We now report that a DAT mutant of hTERT is indeed efficiently rescued upon fusion to hPot1. However, this rescue depended on the ability of hPot1 to localize to telomeres rather than binding to DNA per se. These data support a model whereby the DAT domain of hTERT is implicated in telomere-telomerase associations. 相似文献
5.
Telomeric DNA-binding proteins (TBPs) are crucial components that regulate the structure and function of eukaryotic telomeres and are evolutionarily conserved. We have identified two homologues of AtTBP1 (for Arabidopsis thaliana telomeric DNA binding protein 1), designated as AtTBP2 and AtTRP2, which encode proteins that specifically bind to the telomeric DNA of this plant. These proteins show extensive homology with other known plant TBPs. The isolated C-terminal segments of these proteins were capable of sequence-specific binding to duplex telomeric plant DNA in vitro. DNA bending assays using the Arabidopsis TBPs revealed that AtTBP1 and AtTBP2 have DNA-bending abilities comparable to that of the human homologue hTRF1, and higher than those of AtTRP1 and AtTRP2. 相似文献
6.
Human telomeric repeat binding factor 2 (hTRF2) is a protein that plays an important role in capping human telomeres to protect them from DNA damage repair systems. The ineffectiveness of hTRF2 may be linked to aging and cancer. We report the use of PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) and selective acetylation of lysine residues to study the interaction of the DNA-binding domain and double-stranded telomeric DNA (repeats of TTAGGG). By increasing the resolution of PLIMSTEX to the peptide level, we localized the changes in deuterium uptake of hTRF2 as a function of varying amounts of a model oligodeoxynucleotide. From these experiments, we determined the affinity constant for binding to DNA, which is within a factor of 3 of the previously reported value. Amide H/D exchange revealed portions of the protein that have contacts with the phosphate backbone of DNA, whereas acetylation disclosed the decrease in solvent accessibility of regions containing Lys 447 and 488, which must be involved in interactions with the DNA major and minor grooves. These complementary approaches of amide H/D exchange and selective side chain modification can be employed effectively to pinpoint and quantify protein-ligand, in particular protein-DNA, interactions. 相似文献
7.
Ko S Jun SH Bae H Byun JS Han W Park H Yang SW Park SY Jeon YH Cheong C Kim WT Lee W Cho HS 《Nucleic acids research》2008,36(8):2739-2755
Telomeres are protein–DNA elements that are located at the ends of linear eukaryotic chromosomes. In concert with various telomere-binding proteins, they play an essential role in genome stability. We determined the structure of the DNA-binding domain of NgTRF1, a double-stranded telomere-binding protein of tobacco, using multidimensional NMR spectroscopy and X-ray crystallography. The DNA-binding domain of NgTRF1 contained the Myb-like domain and C-terminal Myb-extension that is characteristic of plant double-stranded telomere-binding proteins. It encompassed amino acids 561–681 (NgTRF1561–681), and was composed of 4 α-helices. We also determined the structure of NgTRF1561–681 bound to plant telomeric DNA. We identified several amino acid residues that interacted directly with DNA, and confirmed their role in the binding of NgTRF1 to telomere using site-directed mutagenesis. Based on a structural comparison of the DNA-binding domains of NgTRF1 and human TRF1 (hTRF1), NgTRF1 has both common and unique DNA-binding properties. Interaction of Myb-like domain with telomeric sequences is almost identical in NgTRF1561–681 with the DNA-binding domain of hTRF1. The interaction of Arg-638 with the telomeric DNA, which is unique in NgTRF1561–681, may provide the structural explanation for the specificity of NgTRF1 to the plant telomere sequences, (TTTAGGG)n. 相似文献
8.
Sue SC Hsiao HH Chung BC Cheng YH Hsueh KL Chen CM Ho CH Huang TH 《Journal of molecular biology》2006,356(1):72-85
The double-stranded telomeric repeat-binding protein (TRP) AtTRP1 is isolated from Arabidopsis thaliana. Using gel retardation assays, we defined the C-terminal 97 amino acid residues, Gln464 to Val560 (AtTRP1(464-560)), as the minimal structured telomeric repeat-binding domain. This region contains a typical Myb DNA-binding motif and a C-terminal extension of 40 amino acid residues. The monomeric AtTRP1(464-560) binds to a 13-mer DNA duplex containing a single repeat of an A.thaliana telomeric DNA sequence (GGTTTAG) in a 1:1 complex, with a K(D) approximately 10(-6)-10(-7) M. Nuclear magnetic resonance (NMR) examination revealed that the solution structure of AtTRP1(464-560) is a novel four-helix tetrahedron rather than the three-helix bundle structure found in typical Myb motifs and other TRPs. Binding of the 13-mer DNA duplex to AtTRP1(464-560) induced significant chemical shift perturbations of protein amide resonances, which suggests that helix 3 (H3) and the flexible loop connecting H3 and H4 are essential for telomeric DNA sequence recognition. Furthermore, similar to that in hTRF1, the N-terminal arm likely contributes to or stabilizes DNA binding. Sequence comparisons suggested that the four-helix structure and the involvement of the loop residues in DNA binding may be features unique to plant TRPs. 相似文献
9.
Targeted deletion reveals an essential function for the telomere length regulator Trf1 总被引:9,自引:0,他引:9
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Karlseder J Kachatrian L Takai H Mercer K Hingorani S Jacks T de Lange T 《Molecular and cellular biology》2003,23(18):6533-6541
The human telomeric DNA binding factor TRF1 (hTRF1) and its interacting proteins TIN2, tankyrase 1 and 2, and PINX1 have been implicated in the regulation of telomerase-dependent telomere length maintenance. Here we show that targeted deletion of exon 1 of the mouse gene encoding Trf1 causes early (day 5 to 6 postcoitus) embryonic lethality. The absence of telomerase did not alter the Terf1(ex1Delta/ex1Delta) lethality, indicating that the phenotype was not due to inappropriate telomere elongation by telomerase. Terf1(ex1Delta/ex1Delta) blastocysts had a severe growth defect of the inner cell mass that was accompanied by apoptosis. However, no evidence was found for telomere uncapping causing this cell death; chromosome spreads of Terf1(ex1Delta/ex1Delta) blastocysts did not reveal chromosome end-to-end fusions, and p53 deficiency only briefly delayed Terf1(ex1Delta/ex1Delta) lethality. These data suggest that murine Trf1 has an essential function that is independent of telomere length regulation. 相似文献
10.
HMf, a histone-related protein from the hyperthermophilic archaeon Methanothermus fervidus, binds preferentially to DNA containing phased tracts of adenines.
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HMf, a histone-related protein from Methanothermus fervidus, was found to bind preferentially to a DNA that is intrinsically bent as a result of the presence of phased oligo(dA) tracts. The intergenic regions in M. fervidus DNA are A+T rich and frequently contain oligo(dA) tracts, some of which may have the size and phasing required to create a net bending in one direction. The binding of HMf to bent DNA could play a direct role in gene expression and stabilization of the genome of this organism. 相似文献
11.
Tzung-Ju Wu Yi-Hsuan Chiang Yi-Chien Lin Chang-Ru Tsai Tai-Yuan Yu Ming-Ta Sung Yan-Hwa Wu Lee Jing-Jer Lin 《The Journal of biological chemistry》2009,284(19):12801-12808
Ku is a heterodimeric protein involved in nonhomologous end-joining of the
DNA double-stranded break repair pathway. It binds to the double-stranded DNA
ends and then activates a series of repair enzymes that join the broken DNA.
In addition to its function in DNA repair, the yeast Saccharomyces
cerevisiae Ku (Yku) is also a component of telomere protein-DNA complexes
that affect telomere function. The yeast telomeres are composed of duplex
C1–3(A/T)G1–3 telomeric DNA repeats plus
single-stranded TG1–3 telomeric DNA tails. Here we show that
Yku is capable of binding to a tailed-duplex DNA formed by telomeric DNA that
mimics the structure of telomeres. Addition of Cdc13p, a single-stranded
telomeric DNA-binding protein, to the Yku-DNA complex enables the formation of
a ternary complex with Cdc13p binding to the single-stranded tail of the DNA
substrate. Because pre-loading of Cdc13p to the single-stranded telomeric tail
inhibits the binding of Yku, the results suggested that loading of Yku and
Cdc13p to telomeres is sequential. Through generating a double-stranded break
near telomeric DNA sequences, we found that Ku protein appears to bind to the
de novo synthesized telomeres earlier than that of Cdc13p in
vivo. Thus, our results indicated that Yku interacts directly with
telomeres and that sequential loading of Yku followed by Cdc13p to telomeres
is required for both proteins to form a ternary complex on telomeres. Our
results also offer a mechanism that the binding of Cdc13p to telomeres might
prevent Yku from initiating DNA double-stranded break repair pathway on
telomeres.DNA damages in the form of double-stranded breaks
(DSBs)4 compromise the
integrity of genomes. Failure in repairing or mis-repairing double-stranded
breaks can lead to chromosome instability and eventually cell death or cancer
(1). Double-stranded breaks are
repaired by two main pathways, the homologous recombination and nonhomologous
DNA end-joining. In nonhomologous DNA end-joining, Ku is the first protein to
bind to the DNA ends to initiate the repair pathway
(2). Upon binding, Ku then
recruits a series of repair enzymes to join the broken ends
(2). Ku is a heterodimeric
protein composed of 70- and ∼80-kDa subunits. In Saccharomyces
cerevisiae, Ku includes Yku70 and Yku80 subunits. Because the biochemical
configuration of the broken ends could be very diverse on DSBs, Ku binds to
double-stranded ends in a sequence- and energy-independent manner. It is
capable of binding to DNA ends with blunt 3′-overhangs or
5′-overhangs as well as double-stranded DNA with nicks, gaps, or
internal loops
(3–7).
However, Ku does not have high affinity to single-stranded DNA. The crystal
structure of human Ku heterodimer indicates that it forms a ring structure
that encircles duplex DNA (7).
This unique structure feature enables Ku to recognize DNA ends and achieves
its high affinity binding.In additional to the role in double-stranded break repair, Ku was shown to
be a component of telomeric protein-DNA complex in yeast and mammals
(8–10).
Telomeres are terminal structures of chromosomes composed of short tandem
repeated sequences (11,
12). Mutation of
YKU70 or YKU80 causes defects in telomere structure
(13–15),
telomere silencing
(16–19),
and replication timing of telomeres
(20). The function of yeast Ku
(Yku) on telomeres could mediate through protein-protein interaction with
Sir4p or protein-RNA interaction with Tlc1 RNA
(21,
22). For example, through the
interaction with Sir4p, Yku selectively affects telomeres silencing but not
the silent mating type loci
(17). Yku could also bind to
telomerase Tlc1 RNA for telomere length maintenance
(22). Judged by the DNA
binding activity of Yku, it is reasonable to suggest that it may bind directly
to telomeric DNA. Indeed, it was shown that human Ku is capable of binding
directly to telomeric DNA in vitro
(15). Moreover, because the
deletion of SIR4 in budding yeast
(23) or Taz1 in
fission yeast (24) does not
abolish the association of Ku with chromosomal ends, this suggests that Ku
might bind directly to telomeric DNA in cells. However, because yeast
telomeres have a short 12–14-mer single-stranded tail
(25), it is uncertain whether
Yku could pass the single-stranded region to reach its binding site. The
direct binding of Yku to telomeric DNA has not been experimentally
determined.In contrast to double-stranded breaks, the ends of linear chromosomes are
not recognized by repair enzymes as DNA damage. In S. cerevisiae,
Cdc13p is the single-stranded TG1–3 DNA-binding protein that
enables cells to differentiate whether the ends of a linear DNA are telomeres
or broken ends
(26–29).
Thus, although the mechanism of how cells prevent the activation of DSB repair
pathway in telomere is unclear, it is likely that binding of Cdc13p to
telomeres might inhibit the initiation of DNA damage response by the Ku
protein. Here, using a tailed-duplex DNA synthesized by telomeric DNA
sequences to mimic telomere structure, we showed that Yku binds directly to
this tailed-duplex DNA substrate and forms a ternary complex with Cdc13p. Our
results also showed that Yku loaded to a de novo synthesized telomere
earlier than Cdc13p in vivo. These results support the direct binding
of Yku to telomeric DNA and that the spatial orientation of Cdc13p might block
the activation of DSB repair pathway on telomeres. 相似文献
12.
The yeast telomere length regulator TEL2 encodes a protein that binds to telomeric DNA. 总被引:4,自引:0,他引:4
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TEL2 is required for telomere length regulation and viability in Saccharomyces cerevisiae. To investigate the mechanism by which Tel2p regulates telomere length, the majority (65%) of the TEL2 ORF was fused to the 3'-end of the gene for maltose binding protein, expressed in bacteria and the purified protein used in DNA binding studies. Rap1p, the major yeast telomere binding protein, recognizes a 13 bp duplex site 5'-GGTGTGTGGGTGT-3' in yeast telomeric DNA with high affinity. Gel shift experiments revealed that the MBP-Tel2p fusion binds the double-stranded yeast telomeric Rap1p site in a sequence-specific manner. Analysis of mutated sites showed that MBP-Tel2p could bind 5'-GTGTGTGG-3' within this 13 bp site. Methylation interference analysis revealed that Tel2p contacts the 5'-terminal guanine in the major groove. MBP-Tel2p did not bind duplex telomeric DNA repeats from vertebrates, Tetrahymena or Oxytricha. These results suggest that Tel2p is a DNA binding protein that recognizes yeast telomeric DNA. 相似文献
13.
The Pot1 (protection of telomeres) protein binds to single-stranded telomeric DNA and is essential for the protection of chromosome ends from degradation and end-to-end fusions. The Pot1 amino-terminal DNA binding domain, Pot1N, adopts an oligonucleotide/oligosaccharide binding fold and binds GGTTAC motifs cooperatively and with exceptionally high sequence specificity. We have now examined DNA binding to naturally occurring telomeric substrates based on the analysis of 100 cloned chromosome ends and in the context of the full-length Pot1 protein. Here, we describe several important differences between Pot1 and Pot1N with apparent consequences for chromosome end protection. Specifically, full-length Pot1.DNA complexes are more stable, and the minimal binding site for a Pot1 monomer is extended into two adjacent telomeric repeats. We provide evidence that Pot1 contains a second DNA binding motif that recognizes DNA with reduced sequence specificity compared with the domain present in Pot1N. The two DNA binding motifs cooperate, whereby the amino-terminal oligonucleotide/oligosaccharide binding fold determines the registry of binding, and the internal DNA binding motif stabilizes the complex and expands the protected region toward the 3' -end. Consistent with a role in chromosome end capping, Pot1 prevents access of telomerase to the 3'-end and protects against exonucleolytic degradation. 相似文献
14.
15.
16.
Human protection of telomeres 1 (POT1) is a negative regulator of telomerase activity in vitro 总被引:10,自引:0,他引:10
The telomeric single-strand DNA binding protein protection of telomeres 1 (POT1) protects telomeres from rapid degradation in Schizosaccharomyces pombe and has been implicated in positive and negative telomere length regulation in humans. Human POT1 appears to interact with telomeres both through direct binding to the 3' overhanging G-strand DNA and through interaction with the TRF1 duplex telomere DNA binding complex. The influence of POT1 on telomerase activity has not been studied at the molecular level. We show here that POT1 negatively effects telomerase activity in vitro. We find that the DNA binding activity of POT1 is required for telomerase inhibition. Furthermore, POT1 is incapable of inhibiting telomeric repeat addition to substrate primers that are defective for POT1 binding, suggesting that in vivo, POT1 likely affects substrate access to telomerase. 相似文献
17.
18.
The herpes simplex virus type 1 origin-binding protein carries out origin specific DNA unwinding and forms stem-loop structures. 总被引:4,自引:1,他引:3
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The UL9 protein of herpes simplex virus type 1 (HSV-1) binds specifically to the HSV-1 oriS and oriL origins of replication, and is a DNA helicase and DNA-dependent NTPase. In this study electron microscopy was used to investigate the binding of UL9 protein to DNA fragments containing oriS. In the absence of ATP, UL9 protein was observed to bind specifically to oriS as a dimer or pair of dimers, which bent the DNA by 35 degrees +/- 15 degrees and 86 degrees +/- 38 degrees respectively, and the DNA was deduced to make a straight line path through the protein complex. In the presence of 4 mM ATP, binding at oriS was enhanced 2-fold, DNA loops or stem-loops were extruded from the UL9 protein complex at oriS, and the DNA in them frequently appeared highly condensed into a tight rod. The stem-loops contained from a few hundred to over one thousand base pairs of DNA and in most, oriS was located at their apex, although in some, oriS was at a border. The DNA in the stem-loops could be stabilized by photocrosslinking, and when Escherichia coli SSB protein was added to the incubations, it bound the stem-loops strongly. Thus the DNA strands in the stem-loops exist in a partially paired, partially single-stranded state presumably making them available for ICP8 binding in vivo. These observations provide direct evidence for an origin specific unwinding by the HSV-1 UL9 protein and for the formation of a relatively stable four-stranded DNA in this process. 相似文献
19.
The ends of linear chromosomes are capped by protein–DNA complexes termed telomeres. Telomere repeat binding factors 1 and 2 (TRF1 and TRF2) bind specifically to duplex telomeric DNA and are critical components of functional telomeres. Consequences of telomere dysfunction include genomic instability, cellular apoptosis or senescence and organismal aging. Mild oxidative stress induces increased erosion and loss of telomeric DNA in human fibroblasts. We performed binding assays to determine whether oxidative DNA damage in telomeric DNA alters the binding activity of TRF1 and TRF2 proteins. Here, we report that a single 8-oxo-guanine lesion in a defined telomeric substrate reduced the percentage of bound TRF1 and TRF2 proteins by at least 50%, compared with undamaged telomeric DNA. More dramatic effects on TRF1 and TRF2 binding were observed with multiple 8-oxo-guanine lesions in the tandem telomeric repeats. Binding was likewise disrupted when certain intermediates of base excision repair were present within the telomeric tract, namely abasic sites or single nucleotide gaps. These studies indicate that oxidative DNA damage may exert deleterious effects on telomeres by disrupting the association of telomere-maintenance proteins TRF1 and TRF2. 相似文献
20.
We present a biochemical analysis of the effects of three single-stranded DNA binding proteins on extension of oligonucleotide primers by the Tetrahymena telomerase. One of them, a human protein designated translin, which was shown to specifically bind the G-rich Tetrahymena and human telomeric repeats, slightly stimulated the primer extension reactions at molar ratios of translin/primer of <1:2. At higher molar ratios, it inhibited the reactions by up to 80%. The inhibition was caused by binding of translin to the primers, rather than by a direct interaction of this protein with telomerase. A second protein, the general human single-stranded DNA binding protein Replication Protein A (RPA), similarly affected the primer extension by telomerase, even though its mode of binding to DNA differs from that of translin. A third protein, the E. coli single-stranded DNA binding protein (SSB), whose binding to DNA is highly cooperative, caused more substantial stimulation and inhibition at the lower and the higher molar ratios of SSB/primer, respectively. Both telomere-specific and general single-stranded DNA binding proteins are found in living cells in telomeric complexes. Based on our data, we propose that these proteins may exert either stimulatory or inhibitory effects on intracellular telomerases, depending on their local concentrations. 相似文献