Rice proteins that bind single-stranded G-rich telomere DNA

In this work, we have identified and characterized proteins in rice nuclear extracts that specifically bind the single-stranded G-rich telomere sequence. Three types of specific DNA-protein complexes (I, II, and III) were identified by gel retardation assays using synthetic telomere substrates consisting of two or more single-stranded TTTAGGG repeats and rice nuclear extracts. Since each complex has a unique biochemical property and differs in electrophoretic mobility, at least three different proteins interact with the G-rich telomere sequences. These proteins are called rice G-rich telomere binding protein (RGBP) and none of them show binding affinity to double-stranded telomere repeats or single-stranded C-rich sequence. Changing one or two G's to C's in the TTTAGGG repeats abolishes binding activity. RGBPs have a greatly reduced affinity for human and Tetrahymena telomeric sequence and do not efficiently bind the cognate G-rich telomere RNA sequence UUUAGGG. Like other telomere binding proteins, RGBPs are resistant to high salt concentrations. RNase sensitivity of the DNA-protein interactions was tested to investigate whether an RNA component mediates the telomeric DNA-protein interaction. In this assay, we observed a novel complex (complex III) in gel retardation assays which did not alter the mobilities or the band intensities of the two pre-existing complexes (I and II). The complex III, in addition to binding to telomeric sequences, has a binding affinity to rice nuclear RNA, whereas two other complexes have a binding affinity to only single-stranded G-rich telomere DNA. Taken together, these studies suggest that RGBPs are new types of telomere-binding proteins that bind in vitro to single-stranded G-rich telomere DNA in the angiosperms.     

Characterization and developmental expression of single-stranded telomeric DNA-binding proteins from mung bean (Vigna radiata)

We have identified and characterized protein factors from mung bean (Vigna radiata) nuclear extracts that specifically bind the single-stranded G-rich telomeric DNA repeats. Nuclear extracts were prepared from three different types of plant tissue, radicle, hypocotyl, and root, in order to examine changes in the expression patterns of telomere-binding proteins during the development of mung bean. At least three types of specific complexes (A, B, and C) were detected by gel retardation assays with synthetic telomere and nuclear extract from radicle tissue, whereas the two major faster-migrating complexes (A and B) were formed with nuclear extracts from hypocotyl and root tissues. Gel retardation assays also revealed differences in relative amount of each complex forming activity in radicle, hypocotyl, and root nuclear extracts. These data suggest that the expression of telomere-binding proteins is developmentally regulated in plants, and that the factor involved in the formation of complex C may be required during the early stages of development. The binding factors have properties of proteins and are hence designated as mung bean G-rich telomere-binding proteins (MGBP). MGBPs bind DNA substrates with three or more single-stranded TTTAGGG repeats, while none of them show binding affinity to either double-stranded or single-stranded C-rich telomeric DNA. These proteins have a lower affinity to human telomeric sequences than to plant telomeric sequences and do not exhibit a significant binding activity to Tetrahymena telomeric sequence or mutated plant telomeric sequences, indicating that their binding activities are specific to plant telomere. Furthermore, RNase treatment of the nuclear extracts did not affect the complex formation activities. This result indicates that the single-stranded telomere-binding activities may be attributed to a simple protein but not a ribonucleoprotein. The ability of MGBPs to bind specifically the single-stranded TTTAGGG repeats may suggest their in vivo functions in the chromosome ends of plants.     

Human Origin Recognition Complex Binds Preferentially to G-quadruplex-preferable RNA and Single-stranded DNA

Origin recognition complex (ORC), consisting of six subunits ORC1–6, is known to bind to replication origins and function in the initiation of DNA replication in eukaryotic cells. In contrast to the fact that Saccharomyces cerevisiae ORC recognizes the replication origin in a sequence-specific manner, metazoan ORC has not exhibited strict sequence-specificity for DNA binding. Here we report that human ORC binds preferentially to G-quadruplex (G4)-preferable G-rich RNA or single-stranded DNA (ssDNA). We mapped the G-rich RNA-binding domain in the ORC1 subunit, in a region adjacent to its ATPase domain. This domain itself has an ability to preferentially recognize G4-preferable sequences of ssDNA. Furthermore, we found, by structure modeling, that the G-rich RNA-binding domain is similar to the N-terminal portion of AdoMet_MTase domain of mammalian DNA methyltransferase 1. Therefore, in contrast with the binding to double-stranded DNA, human ORC has an apparent sequence preference with respect to its RNA/ssDNA binding. Interestingly, this specificity coincides with the common signature present in most of the human replication origins. We expect that our findings provide new insights into the regulations of function and chromatin binding of metazoan ORCs.     

Interaction of an Arabidopsis RNA-binding protein with plant single-stranded telomeric DNA modulates telomerase activity

Telomeres are the specialized structures at the end of linear chromosomes and terminate with a single-stranded 3' overhang of the G-rich strand. The primary role of telomeres is to protect chromosome ends from recombination and fusion and from being recognized as broken DNA ends. This protective function can be achieved through association with specific telomere-binding proteins. Although proteins that bind single-stranded G-rich overhang regulate telomere length and telomerase activity in mammals and lower eukaryotes, equivalent factors have yet to be identified in plants. Here we have identified proteins capable of interacting with the G-rich single-stranded telomeric repeat from the Arabidopsis extracts by affinity chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis indicates that the isolated protein is a chloroplast RNA-binding protein (and a truncated derivative). The truncated derivative, which we refer to as STEP1 (single-stranded telomere-binding protein 1), binds specifically the single-stranded G-rich plant telomeric DNA sequences but not double-stranded telomeric DNA. Unlike the chloroplast-localized full-length RNA-binding protein, STEP1 localizes exclusively to the nucleus, suggesting that it plays a role in plant telomere biogenesis. We also demonstrated that the specific binding of STEP1 to single-stranded telomeric DNA inhibits telomerase-mediated telomere extension. The evidence presented here suggests that STEP1 is a telomere-end binding protein that may contribute to telomere length regulation by capping the ends of chromosomes and thereby repressing telomerase activity in plants.     

Identification of Translin and Trax as Components of the GS1 Strand-Specific DNA Binding Complex Enriched in Brain

Abstract: In previous gel-shift assays, we identified a protein complex, referred to as GS1, that binds in a sequence-specific manner to single-stranded DNA and is highly enriched in brain. As an initial step in clarifying the function of this complex, we have undertaken studies aimed at defining its protein components. In particular, we focused on identifying two protein bands that were covalently labeled when the GS1-DNA complex was subjected to UV irradiation to induce cross-linking between the radiolabeled probe and GS1 components. By following GS1 binding activity through a series of conventional chromatographic steps, as well as an affinity column based on the DNA oligonucleotide used for gel-shift assays, we were able to achieve ∼500,000-fold enrichment of GS1 compared with that in crude cerebellar extracts used as starting material. This highly purified fraction contained both protein bands detected by UV cross-linking in crude extracts. Sequencing of peptides derived from these proteins led to their identification as Translin and Trax, interacting proteins identified in studies of DNA recombination in lymphocytes. A distinct line of research has provided evidence that a complex containing Translin can bind to specific mRNAs and block their translation. Whether one or both of these proposed functions of the Translin/Trax complex explains the high basal level of GS1 binding activity present in the brain remains to be determined.     

The human protein translin specifically binds single-stranded microsatellite repeats, d(GT)n, and G-strand telomeric repeats, d(TTAGGG)n: a study of the binding parameters

We have previously identified in human fibroblasts a multisubunit protein (designated PGB) that specifically bound single-stranded G-rich microsatellite DNA sequences. PGB was later found to be identical, or closely related to translin, an octameric protein that bound single-stranded DNA consisting of sequences flanking chromosomal translocations. Here, we report that recombinant translin binds single-stranded microsatellite repeats, d(GT)n, and G-strand telomeric repeats, d(TTAGGG)n, with higher affinities (Kdis approximately = 2 nM and Kdis approximately = 12.5 nM, respectively, in 100 mM NaCl and 25 degrees C) than the affinity with which it binds a prototypical sequence flanking translocation sites (Kdis approximately = 23 nM). Translin also binds d(GT)n and d(TTAGGG)n overhangs linked to double-stranded DNA with equilibrium constants in the nanomolar range. Formation of DNA quadruplexes by the d(TTAGGG)n repeats inhibits their binding to translin. A further study of the binding parameters revealed that the minimal length of d(GT)n and d(TTAGGG)n oligonucleotides that a translin octamer can bind is 11 nucleotides, but that such oligonucleotides containing up to 30 nucleotides can bind only a single translin octamer. However, the oligonucleotides d(GT)27 and d(TTAGGG)9 bind two octamers with negative cooperativity. Translin does not detectably bind single-stranded d(GT)n sequences embedded within double-stranded DNA. Based on our data, we propose that translin might be involved in the control of recombination at d(GT)n.d(AC)n microsatellites and in telomere maintenance.     

Simian virus 40 (SV40) T antigen binds specifically to double-stranded DNA but not to single-stranded DNA or DNA/RNA hybrids containing the SV40 regulatory sequences.

Simian virus 40 T antigen has been shown previously to bind specifically with high affinity to sites within the regulatory region of double-stranded simian virus 40 DNA. Using competition filter binding and the DNA-binding immunoassay, we show that T antigen did not bind specifically to either early or late single-stranded DNA containing these binding sites. Moreover, T antigen did not bind these sequences present in single-stranded RNA, RNA/RNA duplexes, or RNA/DNA hybrids. T antigen did, however, bind as efficiently to single-stranded DNA-cellulose as to double-stranded DNA-cellulose. This binding was nonspecific because it was independent of the presence of T-antigen-binding sites. The implications of these observations are discussed.     

An explanation for observed estrogen receptor binding to single-stranded estrogen-responsive element DNA.

Estrogen-inducible genes contain an enhancer called the estrogen response element (ERE), a double-stranded inverted repeat. The estrogen receptor (ER) is generally thought to bind to the double-stranded ERE. However, some reports provide evidence that an ER homodimer can bind a single strand of the ERE and suggest that single-stranded ERE binding is the preferred binding mode for ER. Since these two models describe quite different mechanisms of receptor action, we have attempted to reconcile the observations. Analyzing DNA structure by nuclease sensitivity, we found that two identical molecules of a single strand of DNA containing the ERE sequence can partially anneal in an antiparallel manner. Bimolecular annealing produces double-stranded inverted repeats, with adjacent unannealed tails. The amount of annealing correlates exactly with the ability of ER to bind bimolecular EREs. Either strand of an ERE could anneal to itself in a way that would bind ER. We conclude that ER binds only the annealed double-stranded ERE both in vitro and in vivo.     

DRE顺式作用元件dsDNA芯片制备

DRE顺式作用元件能与DREB转录因子特异结合,在诱导逆境（干旱、高盐、低温）基因表达过程中起重要作用。dsDNA（double strand DNA）微阵列芯片技术能够有效地检测序列特异性DNA结合蛋白质（转录因子）与大量DNA靶点（顺式作用元件）的特异性结合,可有效分析生物分子结合作用。根据DRE顺式作用元件核心序列设计并化学合成含发夹结构的单链DNA探针,采用Taq DNA聚合酶在片延伸,并对其在片延伸体系的反应温度、Mg^2＋浓度以及单链探针是否变性等条件进行了优化。结果表明,50％的反应温度,2．5mmol／L的Mg^2＋浓度和单链不变性是TaqDNA聚合酶在片延伸的最佳条件。优化方法制备的dsDNA芯片将更有利于DRE顺式作用元件与DREB抗逆转录因子相互作用的研究。     

DNA recognition and binding by the Euplotes telomere protein.

The 51-kDa telomere protein from Euplotes crassus binds to the extreme terminus of macronuclear telomeres, generating a very salt-stable telomeric DNA-protein complex. The protein recognizes both the sequence and the structure of the telomeric DNA. To explore how the telomere protein recognizes and binds telomeric DNA, we have examined the DNA-binding specificity of the purified protein using oligonucleotides that mimic natural and mutant versions of Euplotes telomeres. The protein binds very specifically to the 3' terminus of single-stranded oligonucleotides with the sequence (T4G4) > or = 3 T4G2; even slight modifications to this sequence reduce binding dramatically. The protein does not bind oligonucleotides corresponding to the complementary C4A4 strand of the telomere or to double-stranded C4A4.T4G4-containing sequences. Digestion of the telomere protein with trypsin generates an N-terminal protease-resistant fragment of approximately 35 kDa. This 35-kDa peptide appears to comprise the DNA-binding domain of the telomere protein as it retains most of the DNA-binding characteristics of the native 51-kDa protein. For example, the 35-kDa peptide remains bound to telomeric DNA in 2 M KCl. Additionally, the peptide binds well to single-stranded oligonucleotides that have the same sequence as the T4G4 strand of native telomeres but binds very poorly to mutant telomeric DNA sequences and double-stranded telomeric DNA. Removal of the C-terminal 15 kDa from the telomere protein does diminish the ability of the protein to bind only to the terminus of a telomeric DNA molecule.