Substrate specificities of bacterial and human AlkB proteins

Methylating agents introduce cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues into nucleic acids, and it was recently demonstrated that the Escherichia coli AlkB protein and two human homologues, hABH2 and hABH3, can remove these lesions from DNA by oxidative demethylation. Moreover, AlkB and hABH3 were also found to remove 1-meA and 3-meC from RNA, suggesting that cellular RNA repair can occur. We have here studied the preference of AlkB, hABH2 and hABH3 for single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and show that AlkB and hABH3 prefer ssDNA, while hABH2 prefers dsDNA. This was consistently observed with three different oligonucleotide substrates, implying that the specificity for single-stranded versus double-stranded DNA is sequence independent. The dsDNA preference of hABH2 was observed only in the presence of magnesium. The activity of the enzymes on single-stranded RNA (ssRNA), double-stranded RNA (dsRNA) and DNA/RNA hybrids was also investigated, and the results generally confirm the notion that while AlkB and hABH3 tend to prefer single-stranded nucleic acids, hABH2 is more active on double-stranded substrates. These results may contribute to identifying the main substrates of bacterial and human AlkB proteins in vivo.     

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N⁶-ethenoadenine (ɛA), 3,N⁴-ethenocytosine (ɛC) and 1,N²-ethenoguanine (1,N²-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate.Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N²-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N²-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins.     

Repair of 3-methylthymine and 1-methylguanine lesions by bacterial and human AlkB proteins

The Escherichia coli AlkB protein repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions in DNA and RNA by oxidative demethylation, a reaction requiring ferrous iron and 2-oxoglutarate as cofactor and co-substrate, respectively. Here, we have studied the activity of AlkB proteins on 3-methylthymine (3-meT) and 1-methylguanine (1-meG), two minor lesions which are structurally analogous to 1-meA and 3-meC. AlkB as well as the human AlkB homologues, hABH2 and hABH3, were all able to demethylate 3-meT in a DNA oligonucleotide containing a single 3-meT residue. Also, 1-meG lesions introduced by chemical methylation of tRNA were efficiently removed by AlkB. Unlike 1-meA and 3-meC, nucleosides or bases corresponding to 1-meG or 3-meT did not stimulate the uncoupled, AlkB-mediated decarboxylation of 2-oxoglutarate. Our data show that 3-meT and 1-meG are repaired by AlkB, but indicate that the recognition of these substrates is different from that in the case of 1-meA and 3-meC.     

Chromatin structure of Drosophila nasutoides satellites as probed by cross-linking DNA in situ

Chromatin structure can be probed by cross-linking DNA in situ using trioxsalen and irradiation with UV light. Presumably DNA within a nucleosome is protected from cross-linking so that this region appears as a single-strand loop in the electron microscope under a condition in which single-strands and double-strands are distinguished. Unprotected regions appear as duplex due to cross-linking.We have used this approach to investigate the structure of chromatins containing satellite DNAs of Drosophila nasutoides. We have previously shown that D. nasutoides has an unusually large autosome pair which is almost entirely heterochromatic. Its nuclear DNA reveals four major satellite components amounting up to 60% of the total genome. All of them are localized in this large heterochromatic chromosome. We wish to ask whether chromatins containing different satellite sequences have different arrangements of nucleosomes. Our results from cross-linking experiments show that all DNA components including main band DNA have different patterns of protected and unprotected regions: (a) The length distributions of protected regions show multiple peaks with the smallest unit lengths being 200 nucleotides for main band DNA, 180 for satellites I, II and III, and 160 for satellite IV. (b) The amounts of unprotected regions, presumably internucleosome DNA, vary from 16% for main band DNA to 60% for satellite IV, suggesting that satellite chromatins have fewer nucleosomes per given length of chromatin than main band DNA chromatin. The spacings between nucleosomes appear to be random in satellite chromatins.     

Covalent cross-linking of proteins without chemical reagents

A facile method for the formation of zero-length covalent cross-links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross-linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85 degrees C for 24 h. Under these conditions, approximately one-third of the total protein present becomes cross-linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero-length cross-links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross-linked dimer has only one amide cross-link and retains the enzymatic activity of the monomer. The in vacuo cross-linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross-linking, and co-lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross-linked dimer.     

Chromium-induced cross-linking of nuclear proteins and DNA

The in vivo cross-linking of proteins to DNA in intact Novikoff ascites hepatoma cells exposed to the chromium salt K2CrO4 was studied. DNA-protein complexes were assayed by high speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and by electrophoretic identification of proteins associated with DNA-containing pellets. Further evidence of DNA-protein complexes, not dissociable in this buffer, was obtained by CsCl gradient centrifugation. Time dependence experiments showed that detectable cross-linking occurred after cells were exposed to chromium salt for at least 4 h, and the amount of DNA-protein complexes increased with longer incubation times. Complex formation occurred only with chromium salt concentrations of 200 microM or greater, and maximal cross-linking was effected at 5 mM. Immunotransfer methodology employing antibodies to nuclear matrix fraction and lamins was used to identify some of the polypeptides comprising the cross-linked complexes. These studies indicated specificity of chromium-induced complex formation within the nuclear protein fractions assayed. Our results document the ability of chromate to produce specific DNA-protein cross-links in living cells.     

Interaction of contractile proteins with DNA

The interaction of contractile proteins (myosin, actin, tropomyosin and troponin) with DNA was studied in vitro using a nitrocellulose filter binding technique. The data indicate a high affinity of myosin and troponin for DNA, a lesser interaction between DNA and tropomyosin and the absence of binding of actin to DNA. When binding to DNA was detected, the interaction was higher with single-stranded DNA than with RNA or double-stranded DNA, although in some conditions myosin binds equally as well to native as to denatured eukaryotic DNA. Myosin binds better to eukaryotic than to phage native DNA.     

Interaction of two new mixed ligand copper(II) complexes with DNA probed by thermodynamic and spectroscopic studies

The DNA binding behavior of [Cu(4,7-dmp)(phen-dione)Cl]Cl (1) and [Cu(2,9-dmp)(phen-dione)Cl]Cl (2) where dmp and phen-dion stand for dimethyl-1,10-phenanthroline and 1,10-phenanthroline-5,6-dion, respectively, was studied with a series of techniques including Viscometry, UV–Vis absorption, circular dichroism and fluorescence spectroscopy. Cytotoxicity effect was also investigated. Thermodynamic parameters, enthalpy and entropy changes were calculated according to Van’t Hoff equation, which indicated that both reactions are predominantly enthalpically driven. However, these two complexes show different behavior in fluorescence, circular dichroism and viscometry methods which indicate the Cu(II) complexes interact with calf-thymus DNA by different mode of binding. These have further been verified by competition studies using Hoechst as a distinct groove binder. All these results indicate that these two complexes (1) and (2) interact with CT-DNA via groove binding and partially intercalative mode, respectively and the binding affinity of the complex 1 is higher than that of complex 2. Finally, our findings suggest that the type of ligands and structure of complexes have marked effect on the binding affinity of complexes involving CT-DNA. Also, these new complexes showed excellent antitumor activity against human T lymphocyte carcinoma-Jurkat cell line.     

Interaction of non-histone proteins HMG1 and HMG2 with core histones in nucleosomes and core particles revealed by chemical cross-linking

Chemical cross-linking was used to study the interaction of the non-histone chromosomal proteins HMG1 and HMG2 with core histones in H1,H5-depleted nucleosomes or core particles. Cross-linking with a 'zero-length' cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and with a longer (cleavable) cross-linker dimethyl-3,3'-dithiobispropionimidate revealed an interaction of HMG1 and HMG2 with (or proximity to) core histones in both types of particles. These results indicated that the presence of the 40-50-base-pairs-long segment of the 'linker' DNA in nucleosomes was not necessary for the establishment of mutual contacts of HMG1 and HMG2 proteins with core histones. Possible implications of the interaction of HMG1 and HMG2 proteins with histones for the structure and functioning of chromatin are discussed.     

Specificity of human trans-sialidase as probed with gangliosides

It has been shown that human blood contains a soluble 67 kDa enzyme, belonging by its donor-acceptor properties to trans-sialidases. The enzyme is capable of both cleaving and synthesizing alpha2-3 and alpha2-6 sialosides [Atherosclerosis2001, 159, 103]. In this work the study of donor-acceptor specificity of the new enzyme was extended. It has been demonstrated in vitro that trans-sialidase possesses the ability of transferring Neu5Ac residue to acceptor (asialofetuin) both from alpha2-3- (GM1, GM3, GD1a), and alpha2-8-sialylated gangliosides (GD3 and GD1b, but not GT1b and GQ1b). Transfer of radiolabeled Neu5Ac from fetuin to glycosphingolipids demonstrated that Lac-Cer>mono- and disialogangliosides>GT1b>GQ1b were acceptors for this enzyme. Two methods were used to reveal whether alpha2-8 bond can be formed between Neu5Ac residues during trans-sialylation, that is immunochemical detection using monoclonal antibodies specific to alpha2-8 di- and oligosialic acids, and fluorometric C7/C9 analysis. Both methods demonstrated the formation of Neu5Acalpha2-8Neu5Ac termination by trans-sialidase, for example, in case of the use 3'SL as sialic acid donor and Neu5Ac-PAA or LDL as acceptor. Thus, human trans-sialidase in vitro displays wide substrate specificity: the enzyme is capable of digesting as well as synthesizing alpha2-3, alpha2-6, and alpha2-8 sialosides.     

Site-specific cross-linking of proteins through tyrosine hexahistidine tags

The genetic addition of hexahistidine (H(6)) tags is widely used to isolate recombinant proteins by immobilized metal-affinity chromatography (IMAC). Addition of a tyrosine residue to H(6) tags enabled proteins to be covalently cross-linked under mild conditions in a manner similar to the natural, site-specific cross-linking of tyrosines into dityrosine. A series of seven hexahistidine tags with tyrosines placed in various positions (H(6)Y tags) were added to the amino terminus of the I28 immunoglobulin domain of the human cardiac titin. The H(6)Y-tagged I28 dimerized in the presence of excess Ni(2+) with a K(D) of 200 microM. Treatment of Ni(2+)-dimerized H(6)Y-I28 with an oxidant, monoperoxyphthalic acid (MMPP) or sodium sulfite, resulted in covalent protein multimerization through chelated Ni(2+)-catalyzed cross-linking of the Y residues engineered into the H(6) tag. The protein oligomerization was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The presence of dityrosine in the cross-linked proteins was confirmed by fluorescence emission at 410 nm. Proteins lacking the Y residue in the H(6) tag treated with the same oxidative conditions did not cross-link or exhibit dityrosine fluorescence, despite the presence of an endogenous Y residue. The method may have potential uses in other protein conjugation applications such as protein labeling and interfacial immobilization of proteins on artificial surfaces.     

Interaction of bacterial lipopolysaccharides with host soluble proteins and polycations

Lipopolysaccharides (LPS) are unique cell wall components of gram-negative bacteria. They represent amphiphilic biopolymeric compounds combining in a single molecule hydrophilic (O-specific chains, core oligosaccharide, etc.) and hydrophobic (lipid A) entities. LPS play a crucial role in various interactions between micro- and macroorganisms and display a broad range of biological activities including toxic activity and ability to activate immune cells. Biological activities of LPS are based on their ability to bind with high affinity to mammalian proteins, e.g., lipoproteins, bactericidal permeability-increasing proteins, lysozyme, etc., and thus to neutralize toxic effects of endotoxins. LPS are specific targets for antimicrobial polycationic compounds used in the therapy of bacterial infections. Studies of mechanisms of toxic effects of LPS culminated in the development of novel approaches to LPS neutralization. One of them is based on the use of compounds able to neutralize LPS toxicity at the expense of formation of macromolecular complexes with them. This approach is highly specific and has no effect on functional activity of antipathogenic defense mechanisms of the host. Interaction of LPS with various classes of cationic amphiphilic molecules including proteins, peptides, and polyamines was the subject of intensive studies in the past decade. Binding of cationic polymers is provided by electrostatic interactions between LPS and negatively charged phosphate and carboxylic groups of LPS localized in lipid A core. The present study is an overview of recently published data on different mechanisms of interactions of LPS with soluble proteins and polycations and modification of physiological activity of LPS.     

Sequence-selective recognition of duplex DNA through covalent interstrand cross-linking: kinetic and molecular modeling studies with pyrrolobenzodiazepine dimers

Members of a homologous series of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers with C8-O-(CH(2))(n)-O-C8' diether linkages (n = 3-6 for 2a-d, respectively) have been studied for their ability to interact with oligonucleotide duplexes containing potential target binding sites. The results confirm earlier predictions that the n = 3 analogue (2a, DSB-120) will covalently bind to a 5'-Pu-GATC-Py sequence by cross-linking opposite-strand guanines separated by 2 bp. Preference for this DNA sequence is shown using oligonucleotides with altered bases between and/or flanking these guanines. The more extended PBD dimer 2c (n = 5) can span an extra base pair and cross-link the 5'-Pu-GA(T/A)TC-Py sequence. The ability of each homologue to cross-link linear plasmid DNA has been determined, with a rank order that correlates with the reported order of in vitro cytotoxicity: n = 3 (2a) > n = 5 (2c) > n = 6 (2d) > n = 4 (2b). The n = 3 homologue (2a) is >300-fold more efficient at cross-linking DNA than the clinically used cross-linking agent melphalan under the same conditions. Kinetic studies reveal that the n = 3 and 5 dimers achieve faster cross-linking to plasmid DNA (108 and 81% cross-linking h(-1) microM(-1) at 37 degrees C, respectively), whereas the n = 4 and 6 homologues are significantly less efficient at 10.3 and 23% cross-linking h(-1) microM(-1), respectively. Alternating activity for the odd n and even n dimers is probably due to configurational factors governed by the spatial separation of the PBD subunits and the flexible character of the tethering linkage. Molecular modeling confirms the order of cross-linking reactivity, and highlights the role of linker length in dictating sequence recognition for this class of DNA-reactive agent.     

Interaction of various nuclear matrix proteins with DNA

An attempt has been made to localize the previously detected strong and weak bonds of nuclear matrix proteins with DNA in some groups of proteins, using fractionation of the matrix into lamina and intranuclear fibrils, isolation of the "elementary globules", fractionation of matrix nucleoproteins on hydroxyapatite. It was shown that both weak and strong bonds are localized on the nuclear lamina and in the intranuclear fibrils. The single-stranded DNA enriched fraction of the matrix nucleoproteins contained mostly strong bonds. The strong bond is less resistant to pronase treatment. A method for isolating nuclear matrix nucleoprotein fractions carrying only strong or only weak bonds is proposed.     

Identification of a 15 kilodalton actin binding region on gizzard caldesmon probed by chemical cross-linking

Fluorescent labeling, limited proteolysis, amino acid sequence determinations, affinity chromatography and specific chemical crosslinking were used to determine the smallest fragment of gizzard caldesmon that interacts with actin. The time course of cleavage with thrombin or submaxillaris arginase-C protease indicates that 90kDa and 35kDa fragments are the two major pieces of the 120kDa native protein. Amino acid sequence determination indicates that the 90kDa fragment is the N-terminal portion of the molecule. Further degradation gave rise to a 15kDa product whose N-terminal amino acid sequence was determined within the first 28 amino acids. Carbodiimide crosslinking with actin revealed that the 15kDa part of the molecule is probably not involved in the actin binding process but may participate in a twisting of the F-actin filament and be responsible of the caldesmon regulatory function during smooth muscle contraction.     

Porphyrin-DNA cross-linking agent hybrids: chemical synthesis and biological studies

Three new porphyrin-DNA cross-linking conjugates 8, 9, and 10 have been synthesized. Their photoinduced DNA cleavage activity have been studied. The IC(50) values to THP-1 cells in the presence of porphyrin derivatives 8, 9, and 10 with photoirradiation were 5.6, 88.4, and 61.8 nM, respectively.     

Tubulin interaction with kinetochore proteins: analysis by in vitro assembly and chemical cross-linking

The sera from patients with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) variation of the autoimmune disease scleroderma contain autoantibodies that specifically recognize the kinetochore by immunofluorescence. Two major antigens of molecular masses 18 and 80 kD are consistently identified by Western blotting of proteins of isolated chromosomes using CREST sera. In this paper, the possible roles that these two proteins play in the interaction of metaphase chromosomes with tubulin and microtubules are examined using two different procedures. In one set of experiments. Chinese hamster ovary (CHO) chromosomes were extracted with 1-2 M NaCl before incubating with phosphocellulose-purified tubulin under in vitro microtubule assembly conditions. After this treatment, the kinetochores of the residual chromosome scaffolds can still initiate the in vitro assembly of microtubules. Immunoblots of the chromosome scaffold proteins demonstrate that the 18-kD protein has been solubilized by the 1-2 M NaCl extraction, suggesting that this protein is not essential for microtubule assembly at the kinetochore. In a second approach, tubulin was covalently cross-linked to kinetochores of CHO chromosomes using the reversible cross-linking reagent dithiobis (succinimidyl propionate). After DNase I digestion, the chromosomes were solubilized and subjected to anti-tubulin affinity chromatography. Tubulin-kinetochore protein complexes were specifically eluted and analyzed by PAGE and immunoblotting with scleroderma CREST serum. Only a small number of proteins were eluted from the antitubulin affinity column as shown by Coomassie Blue-stained gels. In addition to tubulin, an 80-kD polypeptide, bands at 110 and 24 kD, as well as a faint band at 54 kD, can be resolved. Several minor bands can also be seen in silver-stained gels. The 80-kD protein band from whole metaphase chromosomes reacted with scleroderma CREST serum by immunoblotting and therefore probably represents the major centromere antigen CENP-B. This report provides evidence for a specific protein complex on metaphase chromosomes that is contiguous with kinetochore-bound tubulin and may be involved in microtubule-kinetochore interactions during mitosis.     

Three-dimensional interaction of Phi29 pRNA dimer probed by chemical modification interference, cryo-AFM, and cross-linking

Six pRNAs (p for packaging) of bacterial virus phi29 form a hexamer complex that is an essential component of the viral DNA translocating motor. Dimers, the building block of pRNA hexamer, assemble in the order of dimer --> tetramer --> hexamer. The two-dimensional structure of the pRNA monomer has been investigated extensively; however, the three-dimensional structure concerning the distance constraints of the three stems and loops are unknown. In this report, we probed the three-dimensional structure of pRNA monomer and dimer by photo affinity cross-linking with azidophenacyl. Bases 75-81 of the left stem were found to be oriented toward the head loop and proximate to bases 26-31 in a parallel orientation. Chemical modification interference indicates the involvement of bases 45-71 and 82-91 in dimer formation. Dimer was formed via hand-in-hand contact, a novel RNA dimerization that in some aspects is similar to the kissing loops of the human immunodeficiency virus. The covalently linked dimers were found to be biologically active. Both the native dimer and the covalently linked dimer were found by cryo-atomic force microscopy to be similar in global conformation and size.     

Interaction of chromosomal proteins with BrdU substituted DNA as determined by chromatin-DNA competition.

Chromatin-DNA competition has been utilized to examine the general nature of chromosomal proteins interacting more strongly with BrdU substituted DNA. When chromatin is incubated with an excess of purified DNA, a portion of the chromosomal proteins will exchange to the purified DNA. These two complexes can then be separated on Metrizamide gradients due to their differing protein/DNA ratios. Using this technique we observe that most nonhistone chromosomal proteins will exchange to a competitor DNA, the extent of exchange being directly dependent upon the competitor DNA being present in excess. While essentially the same proteins will migrate to either unsubstituted or BrdU substituted DNA, the substituted DNA is found to be a quantitatively better competitor and its effectiveness as a competitor is directly related to the level of BrdU substitution.