Binding of Daunorubicin to β-<scp>d</scp>-Glucosylated DNA Found in Protozoa<i>Tryponosoma brucei</i>Studied by X-ray Crystallography

␤-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 JBP⅐J-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 ϳ15-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.In the DNA of kinetoplastid flagellates, a fraction of thymine is replaced by the modified base ␤-D-glucosyl-hydroxymethyluracil (called base J) 1 (1-3). In all kinetoplastids, J is abundantly present in telomeric repeats (1). In the parasite Trypanosoma brucei, J is also found in the telomeric variant surface glycoprotein (VSG) gene expression sites involved in antigenic variation (4, 5). The presence of J in inactive telomeric VSG gene expression sites but not in the active site suggests that J may be involved in the transcriptional repression of VSG gene expression sites and thus antigenic variation (1, 4 -8).Our discovery of a J-binding protein (JBP) in kinetoplastids that specifically bind J-containing DNA indicates that proteins mediate J function (9). These proteins may then directly or indirectly lead to gene silencing and/or suppression of DNA recombination (6, 10), both of which are involved in the mechanism of antigenic variation. Understanding how JBP specifically recognizes and binds J-DNA may represent a first step in elucidating the function of J and its mechanism of action.We have recently characterized the binding properties of recombinant JBP from Crithidia using synthetic J-DNA substrates that contained the glycosylated base in various sequences and contexts (11). These studies indicated that the JBP/J-DNA interaction is not just simple glucose recognition but rather requires the presentation of the glucose moiety within the major groove of a double-stranded DNA helix. The JBP/J-DNA interaction is not competed by free glucose or free base J, and JBP fails to recognize single-stranded J-DNA or a J-DNA/RN...

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“…Bases for which there is clear evidence for major groove contacts are highlighted in blue and green on the B-form J-DNA structure (26) in Fig. 6.…”

Section: Discussionmentioning

confidence: 99%

Site-specific Interactions of JBP with Base and Sugar Moieties in Duplex J-DNA

Sabatini¹,

Meeuwenoord²,

Boom³

et al. 2002

Journal of Biological Chemistry

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“…This is the case for PDB entry 1da0, the hexanucleotide CGATCG intercalated with daunomycin (Moore et al, 1989), but the same sequence with adriamycin, PDB entry 1d12 (Frederick et al, 1990), fits fully into a combination of B conformers (2ÂBB2, 2B1 and BBB). Another daunomycinintercalated hexamer, PDB entry 308d (Gao et al, 1997), has an unassigned step on one side of its two intercalations, but the other strand uses an unambiguously well fitted BB16, an NtC which also supports glycerol intercalation for the step 312-313 of PDB entry 1mow chain B (Chevalier et al, 2002). Cisplatinum and its derivatives are important DNA-binding anticancer drugs.…”

Section: Annotation Of a Few Selected Prototypical Dna Structuresmentioning

confidence: 99%

A DNA structural alphabet provides new insight into DNA flexibility

Schneider

Božíková

Nečasová

et al. 2018

Acta Cryst Sect D Struct Biol

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DNA is a structurally plastic molecule, and its biological function is enabled by adaptation to its binding partners. To identify the DNA structural polymorphisms that are possible in such adaptations, the dinucleotide structures of 60 000 DNA steps from sequentially nonredundant crystal structures were classified and an automated protocol assigning 44 distinct structural (conformational) classes called NtC (for Nucleotide Conformers) was developed. To further facilitate understanding of the DNA structure, the NtC were assembled into the DNA structural alphabet CANA (Conformational Alphabet of Nucleic Acids) and the projection of CANA onto the graphical representation of the molecular structure was proposed. The NtC classification was used to define a validation score called confal, which quantifies the conformity between an analyzed structure and the geometries of NtC. NtC and CANA assignment were applied to analyze the structural properties of typical DNA structures such as Dickerson-Drew dodecamers, guanine quadruplexes and structural models based on fibre diffraction. NtC, CANA and confal assignment, which is accessible at the website https://dnatco.org, allows the quantitative assessment and validation of DNA structures and their subsequent analysis by means of pseudo-sequence alignment. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Acta_Cryst_D:2.

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“…The crystal structure of J-DNA has indicated that glucose is present in the major groove of B-form DNA (13). The inability of JBP to recognize effectively the RNA:J-DNA duplex may reflect its sensitivity to the global conformation of the helix or the presence of a 2Ј-OH group on the individual sugars interfering with specific protein-nucleic acid backbone interactions.…”

Section: A-form Versus B-form)mentioning

confidence: 99%

Recognition of Base J in Duplex DNA by J-binding Protein

Sabatini¹,

Meeuwenoord²,

Boom³

et al. 2002

Journal of Biological Chemistry

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␤-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 singlestranded 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 DNAbinding protein. The significance of these results in relation to the biological role and mechanism of action of J modification in kinetoplastids is discussed.In the DNA of kinetoplastid flagellates, a fraction of thymine is replaced by the modified base ␤-D-glucosylhydroxymethyluracil, called J (1-3). In all kinetoplastids, J is abundantly present in telomeric repeats (3). In the parasite Trypanosoma brucei, J is also found in the telomeric variant surface glycoprotein (VSG) 1 gene expression sites involved in antigenic variation (4, 5). The presence of J in inactive telomeric VSG gene expression sites but not in the active site suggests that J may be involved in the transcriptional repression of VSG gene expression sites and thus antigenic variation (3-9).It has been suggested that J is involved in long term transcriptional repression and that J could also suppress unwanted recombination between repetitive sequences in the genome (3,8,9). Whether this is true and whether J is the cause or the consequence of this silencing remain to be determined. However, consistent with both ideas, the protrusion of the sugar group of the major groove of DNA, at specific locations, could allow the recognition and binding of proteins that would mediate J function. These proteins could lead to gene silencing and/or suppression of DNA recombination, both of which are involved in the mechanism of antigenic variation. Our recent discovery of J-binding proteins (JBPs) in kinetoplastids that specifically bind J-containing DNA is compatible with this idea (10). By understanding how JBP specifically recognizes and binds the unique modified base in DNA could represent a first step in elucidating the function and mechanism of J action.In this report, we use recombinant JBP to study the interaction between JBP and J-DNA. We utilize DNA ...

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Binding of Daunorubicin to β-d-Glucosylated DNA Found in ProtozoaTryponosoma bruceiStudied by X-ray Crystallography

Cited by 27 publications

References 14 publications

Site-specific Interactions of JBP with Base and Sugar Moieties in Duplex J-DNA

Site-specific Interactions of JBP with Base and Sugar Moieties in Duplex J-DNA

A DNA structural alphabet provides new insight into DNA flexibility

Recognition of Base J in Duplex DNA by J-binding Protein

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