Mechanism of fragility at BCL2 gene minor breakpoint cluster region during t(14;18) chromosomal translocation

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Breaks in chromosome 18 are localized at the 3'-UTR of BCL2 gene or downstream and are mainly clustered in either the major breakpoint region or the minor breakpoint cluster region (mcr). The recombination activating gene (RAG) complex induces breaks at IgH locus of chromosome 14, whereas the mechanism of fragility at BCL2 mcr remains unclear. Here, for the first time, we show that RAGs can nick mcr; however, the mechanism is unique. Three independent nicks of equal efficiency are generated, when both Mg(2+) and Mn(2+) are present, unlike a single nick during V(D)J recombination. Further, we demonstrate that RAG binding and nicking at the mcr are independent of nonamer, whereas a CCACCTCT motif plays a critical role in its fragility, as shown by sequential mutagenesis. More importantly, we recapitulate the BCL2 mcr translocation and find that mcr can undergo synapsis with a standard recombination signal sequence within the cells, in a RAG-dependent manner. Further, mutation to the CCACCTCT motif abolishes recombination within the cells, indicating its vital role. Hence, our data suggest a novel, physiologically relevant, nonamer-independent mechanism of RAG nicking at mcr, which may be important for generation of chromosomal translocations in humans.     

Evidence for a triplex DNA conformation at the bcl-2 major breakpoint region of the t(14;18) translocation

The most common chromosomal translocation in cancer, t(14;18), occurs at the bcl-2 major breakpoint region (Mbr) in follicular lymphomas. The 150-bp bcl-2 Mbr, which contains three breakage hotspots (peaks), has a single-stranded character and, hence, a non-B DNA conformation both in vivo and in vitro. Here, we use gel assays and electron microscopy to show that a triplex-specific antibody binds to the bcl-2 Mbr in vitro. Bisulfite reactivity shows that the non-B DNA structure is favored by, but not dependent upon, supercoiling and suggests a possible triplex conformation at one portion of the Mbr (peak I). We have used circular dichroism to test whether the predicted third strand of that suggested structure can indeed form a triplex with the duplex at peak I, and it does so with 1:1 stoichiometry. Using an intracellular minichromosomal assay, we show that the non-B DNA structure formation is critical for the breakage at the bcl-2 Mbr, because a 3-bp mutation that disrupts the putative peak I triplex also markedly reduces the recombination of the Mbr. A three-dimensional model of such a triplex is consistent with bond length, bond angle, and energetic restrictions (stacking and hydrogen bonding). We infer that an imperfect purine/purine/pyrimidine (R.R.Y) triplex likely forms at the bcl-2 Mbr in vitro, and in vivo recombination data favor this as the major DNA conformation in vivo as well.     

Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3'-UTR of the BCL2 gene and are mainly clustered in the major breakpoint region (MBR). Recently, we found that the BCL2 MBR has a non-B DNA character in genomic DNA. Here, we show that single-stranded DNA modeled from the template strand of the BCL2 MBR, forms secondary structures that migrate faster on native PAGE in the presence of potassium, due to the formation of intramolecular G-quadruplexes. Circular dichroism shows evidence for a parallel orientation for G-quadruplex structures in the template strand of the BCL2 MBR. Mutagenesis and the DMS modification assay confirm the presence of three guanine tetrads in the structure. 1H nuclear magnetic resonance studies further confirm the formation of an intramolecular G-quadruplex and a representative model has been built based on all of the experimental evidence. We also provide data consistent with the possible formation of a G-quadruplex structure at the BCL2 MBR within mammalian cells. In summary, these important features could contribute to the single-stranded character at the BCL2 MBR, thereby contributing to chromosomal fragility.     

Molecular frequency of BCL2/JH t(14; 18) using PCR among lebanese patients with follicular lymphoma: another piece of the geographical map revealed

In follicular lymphoma the frequency of translocation t(14;18) varies considerably across different geographic regions ranging from up to 89% among the American follicular lymphoma to around 30% in the Japanese lymphoma. Neighboring and regional countries varied in their frequency reporting like in Israel (22 of 36 cases; 61%), Turkey (46 of 67 cases; 68.7%), and Jordan (4 of 5 cases; 80%). To our knowledge, this is the first study conducted in Lebanon to determine the frequency of this translocation in follicular lymphoma patients. Of 42 cases diagnosed with follicular lymphoma at the American University of Beirut Medical Center, amplifiable DNA was extracted from the corresponding paraffin embedded tissues and tested for t(14; 18) translocation using PCR amplification of the MBR and MCR breakpoints (INVIVOSCRIBE, CA, USA). We found that 19 patients were positive for t(14; 18) (45.2%) while 23 were negative (54.8%). Among the 19 positive cases, bcl2 was positive in 10 cases (52.6%). The majority of the cases were positive for MBR (40.47%), while only two cases were positive for MCR (4.76%). This study expands the geographical map of the distribution of bcl-2 gene rearrangement in follicular lymphoma patients in the Middle East region. The interesting low frequency of t(14;18) in Lebanese follicular lymphoma patients (45.2%) stands out among several other increased frequencies in surrounding and regional countries. In addition, in this patient population, there is a decreased frequency of the MBR breakpoint (40.47%) while that reported in the literature ranges from 50 to 60%.     

Structure of duplex DNA containing the cisplatin 1,2-{Pt(NH3)2}-d(GpG) cross-link at 1.77 Å resolution

We report the 1.77-Å resolution X-ray crystal structure of a dodecamer DNA duplex with the sequence 5′-CCTCTGGTCTCC-3′ that has been modified to contain a single engineered 1,2-cis-{Pt(NH₃)₂}²⁺-d(GpG) cross-link, the major DNA adduct of cisplatin. These data represent a significant improvement in resolution over the previously published 2.6-Å structure. The ammine ligands in this structure are clearly resolved, leading to improved visualization of the cross-link geometry with respect to both the platinum center and to the nucleobases, which adopt a higher energy conformation. Also better resolved are the deoxyribose sugar puckers, which allow us to re-examine the global structure of platinum-modified DNA. Another new feature of this model is the location of four octahedral [Mg(H₂O)₆]²⁺ ions associated with bases in the DNA major groove and the identification of 124 ordered water molecules that participate in hydrogen-bonding interactions with either the nucleic acid or the diammineplatinum(II) moiety.     

Spectroscopic and structural investigations on [(DD18C6)H2]I8 formed in the reaction of N,N-dibenzylated diazacrown ether (DD18C6) with iodine

Photometric titration measurements indicated in the reaction of diazacrown ether N,N^′-dibenzyl-1,4,10,13-tetraoxo-7,16-diazacyclooctadecane (DD18C6, 2) with iodine in chloroform that a complex was formed in the molar ratio DD18C6:I₂=1:4. This complex was also prepared on a preparative scale as dark brown compound and characterized by microanalysis, UV-Vis, IR, and Raman spectroscopy. By X-ray diffraction analysis the solid-state structure of the complex was shown to be [(DD18C6)H₂]I₈ ([3]I₈) consisting of a doubly protonated macrocycle and an octaiodide dianion (I₈ ²⁻) in the typical (nearly planar) Z-shaped geometry. The macrocycle is C_i symmetric and the protonated nitrogen atoms adopt an endo-endo orientation that is stabilized by the three-center hydrogen bonds N-H(?O)₂, where the oxygen atoms of the macrocycle act as hydrogen acceptors. The orientation of the phenyl groups of the benzyl sidearms are turned above and below the macrocycle. Quantum chemical calculations on the DFT level of theory of the non-protonated and the doubly protonated macrocycle (DD18C6, 2c) and [(DD18C6)H₂]²⁺ (3c), respectively, were performed and discussed for 3c in terms of conformational strain of the macrocycle, the strength of the intramolecular N-H?O hydrogen bonds and cation-π interactions.     

Mono- and dinuclear Fe(III) complexes with the N2O2 donor 5-chlorosalicylideneimine ligands; synthesis, X-ray structural characterization and magnetic properties

Two novel monomeric [C₁₈H₁₇Cl₃N₂O₂Fe] (1) and dimeric [C₃₈H₃₆N₄O₄Cl₆Fe₂] (2) Fe(III) tetradentate Schiff base complexes have been synthesized and their crystal structures have been determined by single crystal X-ray diffraction analysis. In complex (1) the Schiff base ligand coordinates toward one iron atom in a tetradentate mode and each iron atom is five coordinated with the coordination geometry around iron atom which can be described as a distorted square pyramid. The presence of a short (2.89 Å) non-bonding interatomic Fe···O distances between adjacent monomeric Fe(III) complexes results in the formation of a dimer. Structural analysis of compound (2) shows that the structure is a centrosymmetric dimer in which the six coordinated Fe(III) atoms are linked by μ-phenoxo bridges from one of the phenolic oxygen atoms of each Schiff base ligand to the opposite metal center. The variable-temperature (2-300 K) magnetic susceptibility (χ) data of these two compounds have been investigated. The results show that for both complexes Fe(III) centers are in the high spin configuration (S = 5/2) and indicate antiferromagnetic spin-exchange interaction between Fe(III) ions. The obtained results are briefly discussed using magnetostructural correlations developed for other class of iron(III) complexes.     

Stabilization of Mn(II) and Mn(III) in mononuclear complexes derived from tridentate ligands with N2O donors: Synthesis, crystal structure, superoxide dismutase activity and DNA interaction studies

A new family of tridentate ligands PhimpH (2-((2-phenyl-2-(pyridin-2-yl)hydazono)methyl)phenol), N-PhimpH (2-((2-phenyl-2-(pyridin-2-yl)hydrazono)methyl)napthalen-1-ol), Me-PhimpH (2-(1-(2-phenyl-2-(pyridine-2-yl)hydrazono)ethyl)phenol) have been synthesized and characterized. The ligands PhimpH and N-PhimpH after deprotonation react with manganese(II) and manganese(III) starting materials affording [Mn(Phimp)₂] (1), [Mn(Phimp)₂](ClO₄) (2), [Mn(N-Phimp)₂] (3), [Mn(N-Phimp)₂](ClO₄) (4). Complexes [Mn(Phimp)₂] (1) and [Mn(N-Phimp)₂] (3) convert to [Mn(Phimp)₂]⁺ (cation of 2) and [Mn(N-Phimp)₂]⁺ (cation of 4) respectively upon oxidation. Ligand Me-PhimpH stabilized only manganese(III) centre resulting [Mn(Me-Phimp)₂](ClO₄) (5). The molecular structures of [Mn(Phimp)₂], 1 and [Mn(Phimp)₂](ClO₄), 2 were determined by single crystal X-ray diffraction. X-ray crystal structures of 1 and 2 have revealed the presence of distorted octahedral MnN₄O₂ coordination sphere having meridionally spanning ligands. Electrochemical studies for the complexes showed Mn(II)/Mn(III), (E_1/2 = 0.14-0.40 V) and Mn(III)/Mn(IV), (E_1/2 = 0.80-1.06 V) couples vs. Ag/AgCl. The redox properties were exploited to examine superoxide dismutase (SOD) activity using Mn(II)/Mn(III) couple. The complexes 1, 2, 4 and 5 have been revealed to catalyze effectively the dismutation of superoxide () in xanthine-xanthine oxidase-nitro blue tetrazolium assay and IC₅₀ values were found to be 0.29, 0.39, 1.12 and 0.76 μM respectively. DNA interaction studies with complex 2 showed binding of DNA in a non-intercalative pathway. Complexes 1, 2 and 4 exhibited nuclease activity in presence of H₂O₂ and inhibition of activity was noted in presence of KI.     

Effect of the central metal ion on the cleavage of DNA by [M(TPA)Cl]ClO4 complexes (M = Co, Cu and Zn, TPA = tris(2-pyridylmethyl)amine): An efficient artificial nuclease for DNA cleavage

[M(TPA)Cl]ClO₄·nH₂O complexes (1: M = Co^II, n = 0; 2: M = Cu^II, n = ½; 3: M = Zn^II, n = 0) where TPA = tris(2-pyridylmethyl)amine, were synthesized and structurally characterized. The molecular structure of [Cu(TPA)Cl]ClO₄·½H₂O was determined by single crystal X-ray crystallography. In aqueous solution, the complex ions [M(TPA)Cl]⁺ (M = Co^II or Cu^II) are hydrolyzed to the corresponding aqua species [M(TPA)(H₂O)]²⁺. In contrast to the TBP [Cu(TPA)(H₂O)]²⁺, the corresponding TBP cobalt(II) species showed severe distortion towards tetrahedral geometry. The interactions of the three complexes with DNA have been investigated at pH 7.0 (1.0 mM Tris-Cl buffer) and 37 °C. Significant DNA cleavages were obtained for complexes 1 and 2, whereas complex 3 did not show any detectable cleavage for DNA. Under pseudo Michaelis-Menten kinetic conditions, the kinetic parameters k_cat and K_M were determined as k_cat = 6.59 h⁻¹ and K_M = 2.20 × 10⁻⁴ M for 1 and the corresponding parameters for 2 are k_cat = 5.7 × 10⁻² h⁻¹ and K_M = 6.9 × 10⁻⁵ M, and the reactivity of the complexes in promoting the cleavage of DNA decreases in the order 1 > 2 ? 3. The rate enhancements for the DNA cleavage by 1 and 2 correspond to 1.8 × 10⁸ and 1.6 × 10⁶, respectively, over the non-catalyzed DNA. The reactivity of the two complexes was discussed in relation to other related artificial nucleases.     

Macropolyhedral boron-containing cluster chemistry : Products of the reaction between [{Ru(η-MeC6H4Pr)Cl2}2] and syn-[B18H22]. A syn → anti 18-vertex configurational change

Reaction of [{RuCl₂(η⁶-MeC₆H₄^isoPr)}₂] with syn-[B₁₈H₂₂] and non-nucleophilic base results in [8-(η⁶-MeC₆H₄^isoPr)-8-RuB₁₇H₂₁], of 18-vertex anti 10-vertex-nido-10-vertex-nido configuration, as the predominant product. The syn → anti configurational change arises from a trans-cluster pseudo-vertex-substitution of a {BH} vertex by the {Ru₂(η⁶-MeC₆H₄^isoPr)} centre.     

Variation of DNA photocleavage efficiency for [(TL)2Ru(dpp)]Cl2 complexes where TL = 2,2′-bipyridine, 1,10-phenanthroline, or 4,7-diphenyl-1,10-phenanthroline

The complexes [(bpy)₂Ru(dpp)]Cl₂, [(phen)₂Ru(dpp)]Cl₂, and [(Ph₂phen)₂Ru(dpp)]Cl₂ (where dpp = 2,3-bis(2-pyridyl)pyrazine, bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline, Ph₂phen = 4,7-diphenyl-1,10-phenanthroline) have been investigated and found to photocleave DNA via an oxygen-mediated pathway. These light absorbing complexes possess intense metal-to-ligand charge transfer (MLCT) transitions in the visible region of the spectrum. The [(TL)₂Ru(dpp)]²⁺ systems populate ³MLCT states after visible light excitation, giving rise to emissions in aqueous solution centered at 692, 690, and 698 nm for TL = bpy, phen, and Ph₂phen respectively. The ³MLCT states and emissions are quenched by O₂, producing a reactive oxygen species. These complexes photocleave DNA with varying efficiencies, [(Ph₂phen)₂Ru(dpp)]²⁺ > [(phen)₂Ru(dpp)]²⁺ > [(bpy)₂Ru(dpp)]²⁺. The presence of the polyazine bridging ligand will allow these chromophores to be incorporated into larger supramolecular assemblies.     

A new series of iodocarbonyl ruthenium (II) complexes with unsymmetrical phosphine-phosphine sulfide ligands of the type Ph2P(CH2)nP(S)Ph2, n = 1-4: Isolation and structural investigation

Hexa-coordinated chelate complex cis-[Ru(CO)₂I₂(P∩S)] (1a) {P∩S = η²-(P,S)-coordinated} and penta-coordinated non-chelate complexes cis-[Ru(CO)₂I₂(P∼S)] (1b-d) {P∼S = η¹-(P)-coordinated} are produced by the reaction of polymeric [Ru(CO)₂I₂]_n with equimolar quantity of the ligands Ph₂P(CH₂)_nP(S)Ph₂ {n = 1(a), 2(b), 3(c), 4(d)} in dichloromethane at room temperature. The bidentate nature of the ligand a in the complex 1a leads to the formation of five-membered chelate ring which confers extra stability to the complex. On the other hand, 1:2 (Ru:L) molar ratio reaction affords the hexa-coordinated non-chelate complexes cis,cis,trans-[Ru(CO)₂I₂(P∼S)₂] (2a-d) irrespective of the ligands. All the complexes show two equally intense terminal ν(CO) bands in the range 2028-2103 cm⁻¹. The ν(PS) band of complex 1a occurs 23 cm⁻¹ lower region compared to the corresponding free ligand suggesting chelation via metal-sulfur bond formation. X-ray crystallography reveals that the Ru(II) atom occupies the center of a slightly distorted octahedral geometry. The complexes have also been characterized by elemental analysis, ¹H, ¹³C and ³¹P NMR spectroscopy.     

Coexistence of major and minor tautomers of 1-methylcytosine (1-MeC) in a single metal complex, trans-Pt(1-MeC-N3)(1-MeC-N4)X2 (X = Cl, I): metal migration N3 → N4 at acidic pH

Two complexes of composition trans-Pt(1-MeC-N3)(1-MeC-N4)I₂ · 2H₂O (4) and trans-Pt(1-MeC-N3)(1-MeC-N4)Cl₂ (5) are described and characterized by X-ray analysis, which simultaneously contain the preferred aminooxo tautomer I and the rare iminooxo tautomer II of 1-methylcytosine (1-MeC) bonded to the heavy metal, via N3 and N4, respectively. Formation of 4 originates from [Pt(1-MeC-N3)₃I]I (2), which likewise has been characterized by X-ray crystal structure analysis. A feasible way of formation of 4, which involves a metal migration process from N3 to N4 occurring at moderately acidic pH, is proposed. It appears to be yet another mechanism of metal migration, different from previously established cases which are redox-assisted and hydroxide-promoted, respectively.