Site-specific interactions of JBP with base and sugar moieties in duplex J-DNA. Evidence for both major and minor groove contacts

Beta-D-Glucosyl-hydroxymethyluracil, also called base J, is an unusually modified DNA base conserved among Kinetoplastida. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes. We have previously identified a J-binding protein (JBP) in Trypanosoma, Leishmania, and Crithidia, and we have shown that it is a structure-specific binding protein. Here we examine the molecular interactions that contribute to recognition of the glycosylated base in synthetic DNA substrates using modification interference, modification protection, DNA footprinting, and photocross-linking techniques. We find that the two primary requirements for J-DNA recognition include contacts at base J and a base immediately 5' of J (J-1). Methylation interference analysis indicates that the requirement of the base at position J-1 is due to a major groove contact independent of the sequence. DNA footprinting of the JBP.J-DNA complex with 1,10-phenanthroline-copper demonstrates that JBP contacts the minor groove at base J. Substitution of the thymine moiety of J with cytosine reduces the affinity for JBP approximately 15-fold. These data indicate that the sole sequence dependence for JBP binding may lie in the thymine moiety of base J and that recognition requires only two specific base contacts, base J and J-1, within both the major and minor groove of the J-DNA duplex.     

The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase

Trypanosomatids contain an unusual DNA base J (beta-d-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe(2+) and 2-oxoglutarate-dependent dioxygenases and that replacement of conserved residues putatively involved in Fe(2+) and 2-oxoglutarate-binding inactivates the ability of JBP1 to contribute to J synthesis without affecting its ability to bind to J-DNA. We propose that JBP1 is a thymidine hydroxylase responsible for the local amplification of J inserted by JBP2, another putative thymidine hydroxylase.     

Regulation of trypanosome DNA glycosylation by a SWI2/SNF2-like protein

Synthesis of the modified thymine base beta-D-glucosyl-hydroxymethyluracil, or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream-form-specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. The mechanism of developmental and telomeric-specific regulation of J synthesis is unknown. We have previously identified a J binding protein (JBP1) involved in propagating J synthesis. We have now identified a homolog of JBP1, JBP2, containing a domain related to the SWI2/SNF2 family of chromatin remodeling proteins that is upregulated in bloodstream form cells and interacts with nuclear chromatin. We show that expression of JBP2 in procyclic form cells leads to de novo J synthesis within telomeric regions of the chromosome and that this activity is inhibited after mutagenesis of conserved residues critical for SWI2/SNF2 function. We propose a model in which chromatin remodeling by JBP2 regulates the initial sites of J synthesis within bloodstream form trypanosome DNA, with further propagation and maintenance of J by JBP1.     

Formation of linear inverted repeat amplicons following targeting of an essential gene in Leishmania

Attempts to inactivate an essential gene in the protozoan parasite Leishmania have often led to the generation of extra copies of the wild-type alleles of the gene. In experiments with Leishmania tarentolae set up to disrupt the gene encoding the J-binding protein 1 (JBP1), a protein binding to the unusual base beta-D-glucosyl-hydroxymethyluracil (J) of Leishmania, we obtained JBP1 mutants containing linear DNA elements (amplicons) of approximately 100 kb. These amplicons consist of a long inverted repeat with telomeric repeats at both ends and contain either the two different targeting cassettes used to inactivate JBP1, or one cassette and one JBP1 gene. Each long repeat within the linear amplicons corresponds to sequences covering the JBP1 locus, starting at the telomeres upstream of JBP1 and ending in a approximately 220 bp sequence repeated in an inverted (palindromic) orientation downstream of the JBP1 locus. We propose that these amplicons have arisen by a template switch inside a DNA replication fork involving the inverted DNA repeats and helped by the gene targeting.     

J-binding protein increases the level and retention of the unusual base J in trypanosome DNA

The nuclear DNA of Trypanosoma brucei and other kinetoplastid flagellates contains the unusual base beta-d-glucosyl-hydroxymethyluracil, called J, replacing part of the thymine in repetitive sequences. We have described a 100 kDa protein that specifically binds to J in duplex DNA. We have now disrupted the genes for this J-binding protein (JBP) in T. brucei. The disruption does not affect growth, gene expression or the stability of some repetitive DNA sequences. Unexpectedly, however, the JBP KO trypanosomes contain only about 5% of the wild-type level of J in their DNA. Excess J, randomly introduced into T. brucei DNA by growing the cells in the presence of the J precursor 5-hydroxymethyldeoxyuridine, is lost by simple dilution as the KO trypanosomes multiply, showing that JBP does not protect J against removal. In contrast, cells containing JBP lose excess J only sluggishly. We conclude that JBP is able to activate the thymine modification enzymes to introduce additional J in regions of DNA already containing a basal level of J. We propose that JBP is a novel DNA modification maintenance protein.     

The structural basis for recognition of base J containing DNA by a novel DNA binding domain in JBP1

The J-binding protein 1 (JBP1) is essential for biosynthesis and maintenance of DNA base-J (β-d-glucosyl-hydroxymethyluracil). Base-J and JBP1 are confined to some pathogenic protozoa and are absent from higher eukaryotes, prokaryotes and viruses. We show that JBP1 recognizes J-containing DNA (J-DNA) through a 160-residue domain, DB-JBP1, with 10 000-fold preference over normal DNA. The crystal structure of DB-JBP1 revealed a helix-turn-helix variant fold, a ‘helical bouquet’ with a ‘ribbon’ helix encompassing the amino acids responsible for DNA binding. Mutation of a single residue (Asp525) in the ribbon helix abrogates specificity toward J-DNA. The same mutation renders JBP1 unable to rescue the targeted deletion of endogenous JBP1 genes in Leishmania and changes its distribution in the nucleus. Based on mutational analysis and hydrogen/deuterium-exchange mass-spectrometry data, a model of JBP1 bound to J-DNA was constructed and validated by small-angle X-ray scattering data. Our results open new possibilities for targeted prevention of J-DNA recognition as a therapeutic intervention for parasitic diseases.     

The modified base J is the target for a novel DNA-binding protein in kinetoplastid protozoans

DNA from Kinetoplastida contains the unusual modified base beta-D-glucosyl(hydroxymethyl)uracil, called J. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes in Trypanosoma brucei. We have now identified a protein in nuclear extracts of bloodstream stage T.brucei that binds specifically to J-containing duplex DNA. J-specific DNA binding was also observed with extracts from the kinetoplastids Crithidia fasciculata and Leishmania tarentolae. We purified the 90 kDa C.fasciculata J-binding protein 50 000-fold and cloned the corresponding gene from C.fasciculata, T.brucei and L.tarentolae. Recombinant proteins expressed in Escherichia coli demonstrated J-specific DNA binding. The J-binding proteins show 43-63% identity and are unlike any known protein. The discovery of a J-binding protein suggests that J, like methylated cytosine in higher eukaryotes, functions via a protein intermediate.     

Universal minicircle sequence-binding protein, a sequence-specific DNA-binding protein that recognizes the two replication origins of the kinetoplast DNA minicircle

Replication of the kinetoplast DNA minicircle lagging (heavy (H))-strand initiates at, or near, a unique hexameric sequence (5'-ACGCCC-3') that is conserved in the minicircles of trypanosomatid species. A protein from the trypanosomatid Crithidia fasciculata binds specifically a 14-mer sequence, consisting of the complementary strand hexamer and eight flanking nucleotides at the H-strand replication origin. This protein was identified as the previously described universal minicircle sequence (UMS)-binding protein (UMSBP) (Tzfati, Y., Abeliovich, H., Avrahami, D., and Shlomai, J. (1995) J. Biol. Chem. 270, 21339-21345). This CCHC-type zinc finger protein binds the single-stranded form of both the 12-mer (UMS) and 14-mer sequences, at the replication origins of the minicircle L-strand and H-strand, respectively. The attribution of the two different DNA binding activities to the same protein relies on their co-purification from C. fasciculata cell extracts and on the high affinity of recombinant UMSBP to the two origin-associated sequences. Both the conserved H-strand hexamer and its flanking nucleotides at the replication origin are required for binding. Neither the hexameric sequence per se nor this sequence flanked by different sequences could support the generation of specific nucleoprotein complexes. Stoichiometry analysis indicates that each UMSBP molecule binds either of the two origin-associated sequences in the nucleoprotein complex but not both simultaneously.     

DNA Sequence Determinants Controlling Affinity,Stability and Shape of DNA Complexes Bound by the Nucleoid Protein Fis

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.     

Preferred binding sites for the bifunctional intercalator TANDEM determined using DNA fragments that contain every symmetrical hexanucleotide sequence

We have prepared novel DNA footprinting substrates that contain all 64 symmetrical hexanucleotide sequences. These were contained in two restriction fragments that were cloned into the pUC19 polylinker site; each fragment was also obtained in both orientations. These fragments were used to assess the sequence binding preferences of the synthetic quinoxaline antibiotic TANDEM. We found that, although the ligand binds to most TpA steps, the affinity is affected by the flanking sequences. The best binding sites contain the tetranucleotide sequence ATAT, although YATATR is a better site than RATATY. TTAA always is a poor binding site, especially TTTAAA. The binding to GTAC is strongly dependent on the flanking bases, with good binding to GGTACC but none at all to CGTACG.     

Efficient binding of glucocorticoid receptor to its responsive element requires a dimer and DNA flanking sequences

A combination of the gel retardation assay and interference by hydroxyl radical modification (missing nucleoside technique) was used to analyze the interaction of the glucocorticoid receptor (GR) with various glucocorticoid responsive elements (GRE). Short oligonucleotides containing the 15-bp GRE and 1 to 3 flanking base pairs on each side, are bound with very low affinity. The same GREs, when positioned in the center of a large DNA fragment (40-50 bp), show high affinity for the receptor. However, when the GRE is positioned at the border of a 54-bp fragment, the affinity of the GR for the GRE decreases markedly. The DNA binding affinity increases linearly with each added flanking base pair and optimal binding is observed with 8-10 flanking bp. Thus, the nonconserved DNA sequences flanking the GRE contribute significantly to the free energy of receptor binding to DNA. Using larger DNA fragments (greater than 100 bp) and a smaller form of the receptor (40 kD), two retarded complexes are found that correspond to monomeric and homodimeric receptor DNA complexes. The DNA-binding domain of the GR (20 kD), expressed in bacteria, binds to the GRE as a monomer as well as a dimer and can form heterodimers with the native 94-kD GR. Insertion or deletion of one single base pair between the two halves of the GRE reduces the affinity for the homodimeric form of the native GR, and inhibits the function of the GRE in gene transfer experiments, suggesting that a dimer of the GR is the functional entity that binds to the GRE.     

DNA sequence recognition by an isopropyl substituted thiazole polyamide

We have used DNA footprinting and fluorescence melting experiments to study the sequence-specific binding of a novel minor groove binding ligand (thiazotropsin A), containing an isopropyl substituted thiazole polyamide, to DNA. In one fragment, which contains every tetranucleotide sequence, sub-micromolar concentrations of the ligand generate a single footprint at the sequence ACTAGT. This sequence preference is confirmed in melting experiments with fluorescently labelled oligonucleotides. Experiments with DNA fragments that contain variants of this sequence suggest that the ligand also binds, with slightly lower affinity, to sequences of the type XCYRGZ, where X is any base except C, and Z is any base except G.     

A CT promoter element binding protein: definition of a double-strand and a novel single-strand DNA binding motif.

Numerous genes contain promoter elements that are nuclease hypersensitive. These elements frequently possess polypurine/polypyrimidine stretches and are usually associated with altered chromatin structure. We have previously isolated a clone that binds a class of CT-rich promoter elements. We have further characterized this clone, termed the nuclease-sensitive element protein-1, or NSEP-1. NSEP-1 binds both duplex CT elements and the CT-rich strand of these elements in a 'generic' sequence specific manner and has overlapping but distinct single-and double-strand DNA binding domains. The minimal peptide region sufficient for both duplex and single-strand DNA binding includes two regions rich in basic amino acids flanking an RNP-CS-1 like octapeptide motif. Deletion analysis shows that the single-strand DNA binding activity is mediated by the RNP-CS-1 like octapeptide motif and is the key peptide region necessary for single-strand binding. NSEP-1's affinity for CT rich promoter elements with strand asymmetry in addition to its double- and single-strand DNA binding properties suggests that it may be a member of a class of DNA binding proteins that modulate gene expression by their ability to recognize DNA with unusual secondary structure.     

Human Rap1 Interacts Directly with Telomeric DNA and Regulates TRF2 Localization at the Telomere

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.     

Identification and characterization of a putative telomere end-binding protein from Tetrahymena thermophila.

Telomeric DNA of Tetrahymena thermophila consists of a long stretch of (TTGGGG)n double-stranded repeats with a single-stranded (TTGGGG)2 3' overhang at the end of the chromosome. We have identified and characterized a protein that specifically binds to a synthetic telomeric substrate consisting of duplex DNA and the 3' telomeric repeat overhang. This protein is called TEP (telomere end-binding protein). A change from G to A in the third position of the TTGGGG overhang repeat converts the substrate to a human telomere analog and reduces the binding affinity approximately threefold. Changing two G's to C's in the TTGGGG repeats totally abolishes binding. However, permutation of the Tetrahymena repeat sequence has only a minor effect on binding. A duplex structure adjacent to the 3' overhang is required for binding, although the duplex need not contain telomeric repeats. TEP does not bind to G-quartet DNA, which is formed by many G-rich sequences. TEP has a greatly reduced affinity for RNA substrates. The copy number of TEP is at least 2 x 10(4) per cell, and it is present under different conditions of cell growth and development, although its level varies. UV cross-linking experiments show that TEP has an apparent molecular mass of approximately 65 kDa. Unlike other telomere end-binding proteins, TEP is sensitive to high salt concentrations.