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.     

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.     

Identification of the Glucosyltransferase That Converts Hydroxymethyluracil to Base J in the Trypanosomatid Genome

O-linked glucosylation of thymine in DNA (base J) is an important regulatory epigenetic mark in trypanosomatids. β-d-glucopyranosyloxymethyluracil (base J) synthesis is initiated by the JBP1/2 enzymes that hydroxylate thymine, forming 5-hydroxymethyluracil (hmU). hmU is then glucosylated by a previously unknown glucosyltransferase. A recent computational screen identified a possible candidate for the base J-associated glucosyltransferase (JGT) in trypanosomatid genomes. We demonstrate that recombinant JGT utilizes uridine diphosphoglucose to transfer glucose to hmU in the context of dsDNA. Mutation of conserved residues typically involved in glucosyltransferase catalysis impairs DNA glucosylation in vitro. The deletion of both alleles of JGT from the genome of Trypanosoma brucei generates a cell line that completely lacks base J. Reintroduction of JGT in the JGT KO restores J synthesis. Ablation of JGT mRNA levels by RNAi leads to the sequential reduction in base J and increased levels of hmU that dissipate rapidly. The analysis of JGT function confirms the two-step J synthesis model and demonstrates that JGT is the only glucosyltransferase enzyme required for the second step of the pathway. Similar to the activity of the related Ten-Eleven Translocation (TET) family of dioxygenases on 5mC, our studies also suggest the ability of the base J-binding protein enzymes to catalyze iterative oxidation of thymine in trypanosome DNA. Here we discuss the regulation of hmU and base J formation in the trypanosome genome by JGT and base J-binding protein.     

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.     

Recognition of base J in duplex DNA by J-binding protein.

beta-d-Glucosylhydroxymethyluracil, also called base J, is an unusual 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 found a J-binding protein (JBP) in Trypanosoma, Leishmania, and Crithidia. We have now characterized the binding properties of recombinant JBP from Crithidia using synthetic J-DNA substrates that contain the glycosylated base in various DNA sequences. We find that JBP recognizes base J only when presented in double-stranded DNA but not in single-stranded DNA or in an RNA:DNA duplex. It also fails to interact with free glucose or free base J. JBP is unable to recognize nonmodified DNA or intermediates of J synthesis, suggesting that JBP is not directly involved in J biosynthesis. JBP binds J-DNA with high affinity (K(d) = 40-140 nm) but requires at least 5 bp flanking the glycosylated base for optimal binding. The nature of the flanking sequence affects binding because J in a telomeric sequence binds JBP with higher affinity than J in another sequence known to contain J in trypanosome DNA. We conclude that JBP is a structure-specific DNA-binding protein. The significance of these results in relation to the biological role and mechanism of action of J modification in kinetoplastids is discussed.     

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.     

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.     

Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids

Modified bases in nucleic acids present a layer of information that directs biological function over and beyond the coding capacity of the conventional bases. While a large number of modified bases have been identified, many of the enzymes generating them still remain to be discovered. Recently, members of the 2-oxoglutarate- and iron(II)-dependent dioxygenase superfamily, which modify diverse substrates from small molecules to biopolymers, were predicted and subsequently confirmed to catalyze oxidative modification of bases in nucleic acids. Of these, two distinct families, namely the AlkB and the kinetoplastid base J binding proteins (JBP) catalyze in situ hydroxylation of bases in nucleic acids. Using sensitive computational analysis of sequences, structures and contextual information from genomic structure and protein domain architectures, we report five distinct families of 2-oxoglutarate- and iron(II)-dependent dioxygenase that we predict to be involved in nucleic acid modifications. Among the DNA-modifying families, we show that the dioxygenase domains of the kinetoplastid base J-binding proteins belong to a larger family that includes the Tet proteins, prototyped by the human oncogene Tet1, and proteins from basidiomycete fungi, chlorophyte algae, heterolobosean amoeboflagellates and bacteriophages. We present evidence that some of these proteins are likely to be involved in oxidative modification of the 5-methyl group of cytosine leading to the formation of 5-hydroxymethylcytosine. The Tet/JBP homologs from basidiomycete fungi such as Laccaria and Coprinopsis show large lineage-specific expansions and a tight linkage with genes encoding a novel and distinct family of predicted transposases, and a member of the Maelstrom-like HMG family. We propose that these fungal members are part of a mobile transposon. To the best of our knowledge, this is the first report of a eukaryotic transposable element that encodes its own DNA-modification enzyme with a potential regulatory role. Through a wider analysis of other poorly characterized DNA-modifying enzymes we also show that the phage Mu Mom-like proteins, which catalyze the N6-carbamoylmethylation of adenines, are also linked to diverse families of bacterial transposases, suggesting that DNA modification by transposable elements might have a more general presence than previously appreciated. Among the other families of 2-oxoglutarate- and iron(II)-dependent dioxygenases identified in this study, one which is found in algae, is predicted to mainly comprise of RNA-modifying enzymes and shows a striking diversity in protein domain architectures suggesting the presence of RNA modifications with possibly unique adaptive roles. The results presented here are likely to provide the means for future investigation of unexpected epigenetic modifications, such as hydroxymethyl cytosine, that could profoundly impact our understanding of gene regulation and processes such as DNA demethylation.     

An effective method for the in situ synthesis of DNA-CPG conjugates using chemical ligation technology as tools for SNP analysis

In this paper, we report a new method for the SNP analysis by using a chemical ligation (CL) technique on CPG plates with high coupling efficiency. This method showed markedly high match/mismatch discrimination ability. Particularly, replacement of thymidine with 2-thiothymidine in DNA probes used in the CL technology resulted in significant improvement of the base discrimination ability of the thymine base in this system.     

Thymine metabolism in Pseudomonas aeruginosa strain 1: the presence of a salvage pathway

Exogenous thymine was found to be taken up very slowly by Pseudomonas aeruginosa in comparison to other pyrimidines, and most of it was catabolized by the cell. The existence of a functional, although inefficient, thymine salvage pathway was demonstrated and this pathway operated more effectively when de novo thymidine nucleotide biosynthesis was inhibited by trimethoprim or methotrexate. The mechanism of thymine salvage by P. aeruginosa appears to be different from that of Escherichia coli and Pseudomonas acidovorans as thymidine was not incorporated into the DNA. Like P. acidovorans, P. aeruginosa lacked thymidine phosphorylase activity. Unsuccessful attempts were made to isolate thymine auxotrophs.     

NADH-dependent O-deethylation of p-nitrophenetole with rabbit liver microsomes.

A previous paper in this series (C. K. Mathews, (1972) J. Biol. Chem.247, 7430) showed that deoxynucleoside triphosphate pools expand manyfold when DNA synthesis is blocked genetically in infection by bacteriophage T4. This paper describes a more detailed analysis of this phenomenon. The key approach involves labeling with thymine or thymidine under conditions of infection where both phage and host bear mutations that inactivate thymidylate synthetase. Principal findings include the following: (1) Nucleotides in the expanded pools are derived in roughly equal measure from breakdown of host cell DNA and from nucleotide synthesis de novo after infection. (2) Thymidine diphosphate pool expansion is comparable, in rate and extent, to thymidine triphosphate pool expansion, but thymidine monophosphate pools accumulate much less. (3) The rate of expansion of the total thymine nucleotide pool following temperature upshift in infection by a temperature-sensitive gene 45 mutant is approximately equal to the rate of thymine incorporation into DNA immediately preceding the upshift. (4) Similarly, when DNA synthesis is restored by a downshift, the total thymine nucleotide pool drains at a rate commensurate with that of thymine incorporation into DNA. (5) Under these latter conditions the dTTP pool begins to drain earlier than the dTDP pool, suggesting that dTTP is the more proximal DNA precursor in this system.     

Interaction of caffeine with the DNA of Chinese hamster cells.

Incubation of Chinese hamster cells with labelled caffeine leads to transfer of radioactivity to DNA. This association occurs during the S phase of the cell cycle and involves parental as well as newly synthesised strands. The replacement of thymidine by BrdUrd prevents the incorporation radioactivity from caffeine into the DNA strands containing BrdUrd. Thymine is the only base which becomes labelled and data suggesting the participation of methyl groups of caffeine in the biosynthesis of thymine are presented. Ultraviolet irradiation increases the incorporation of radioactivity from caffeine to DNA.     

Pyrimidine deoxyribonucleotide metabolism in Acholeplasma laidlawii B-PG9.

Extracts of Acholeplasma laidlawii B-PG9 were examined for the enzymes associated with the interconversion of the pyrimidine deoxyribonucleotides and the biosynthesis of thymidine nucleotides. A. laidlawii B-PG9 possessed deaminases for deoxycytidine and dCMP, pyrophosphatases for dUTP, phosphorylases for thymidine and uridine, and a membrane-associated pyrimidine deoxyribonucleoside monophosphate phosphatase activity. The role these enzyme activities have in the generation of deoxyribose-1-phosphate during growth may explain the ability of A. laidlawii B-PG9 to utilize either thymine or thymidine for biosynthesis.     

The association between prolyl hydroxylase metabolism and cell growth in cultured L-929 fibroblasts

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis, its active form is a tetramer (alpha 2 beta 2). In L-929 fibroblasts in the log phase of culture there is a low level of active enzyme. When the cell culture reaches confluency, prolyl hydroxylase activity in cells increases by a process that requires de novo RNA and protein synthesis. The same result may be achieved by crowding the cells (replating log phase cells at the density of stationary phase cells). In the work reported here we further examined induction of the enzyme. RNA synthesis necessary for enzyme induction is complete 6 h after "crowding" while protein synthesis requires 12 h. Thymidine (0.2-0.5 mM) added to log phase cells will also cause enzyme induction to the level found in "crowded" or resting cells. We also looked at the decay of the enzyme activity after subculture. This occurs rapidly (enzyme half-life is 1-2 h) and is concurrent with the re-entry of resting cells into cell cycle; however, thymidine added at the time of subculture to block DNA synthesis does not prevent the loss of prolyl hydroxylase activity. These results suggest that when cells are not engaged in propagation, they begin to synthesize luxury proteins such as prolyl hydroxylase. However, the loss of prolyl hydroxylase during subculture is probably not a direct consequence of DNA synthesis.