Linkage structures strongly influence the binding cooperativity of DNA intercalators conjugated to triplex forming oligonucleotides.

Conjugation of DNA intercalators to triple helix forming oligodeoxynucleotides (ODN's) can enhance ODN binding properties and consequently their potential ability to modulate gene expression. To test the hypothesis that linkage structure could strongly influence the binding enhancement of intercalator conjugation with triplex forming ODN's, we have used a model system to investigate binding avidity of short oligomers conjugated to DNA intercalators through various linkages. Using a dA10.T10 target sequence imbedded in a 20 bp duplex, binding avidities of a T10 ODN joined to the DNA intercalator 6,9-diamino, 3-methoxy acridine (DAMA) by 8 different 5' linkages were measured using an electrophoretic mobility shift assay. Although unmodified T10 has a very limited capacity for stable binding under these conditions (apparent Kd > 250 microM at 4 degrees C), conjugation to DAMA using flexible linkers of certain lengths and chemical compositions greatly enhanced binding (Kd of 1 microM at 4 degrees C). Other linkers, however, modestly enhanced binding or had no effect on binding at all. Thus, the length, flexibility, and chemical composition of linker structures all substantially influence intercalator conjugated oligodeoxynucleotide binding avidity.     

Initial steps in Haemophilus influenzae transformation. Donor DNA binding in the com10 mutant

The com10 mutant of Haemophilus influenzae binds donor DNA reversibly, but is deficient in uptake. The DNA binding has all the characteristics of interaction with a protein receptor; it is saturable, reversible, and specific. However, binding specificity is 6-fold weaker in com10 than is uptake specificity in wild-type. The binding of small (120 base pairs) and large (14,400 base pairs) DNA molecules were compared. For small molecules, binding data fitted a straight line by Scatchard analysis (Bmax = 4.8 DNA molecules/cell, Kd = 0.5 X 10(-9) M). In contrast, for large DNA molecules, the Scatchard plot was not linear. A high affinity binding (Kd = 0.4 X 10(-12) M) and a lower affinity binding (Kd = 1.2 X 10(-11) M) were found with a total number of 3 molecules bound per cell. In wild-type cells, 3.2 large molecules were taken up per cell, whereas up to 40 small 120-base pair DNA fragments were taken up per cell. Uptake of small DNA molecules followed a Michaelis-Menten function with a Km of 0.5 X 10(-9) M and a maximal initial velocity of 1.5 molecules/cell/min at room temperature. For large DNA molecules, maximal initial velocity was approximately 2 molecules/cell/min at room temperature. The analysis of the binding and uptake data suggest to us that a receptor or a receptor complex is responsible for the uptake of either a single large DNA molecule or, successively, a number of small DNA molecules.     

Sequence-specific DNA binding by the MspI DNA methyltransferase.

The MspI methyltransferase (M.MspI) recognizes the sequence CCGG and catalyzes the formation of 5-methylcytosine at the fist C-residue. We have investigated the sequence-specific DNA-binding properties of M.MspI under equilibrium conditions, using gel-mobility shift assays and DNasel footprinting. M.MspI binds to DNA in a sequence-specific manner either alone or in the presence of the normal methyl donor S-adenosyl-L-methionine as well as the analogues, sinefungin and S-adenosyl-L-homocysteine. In the presence of S-adenosyl-L-homocysteine, M.MspI shows the highest binding affinity to DNA containing a hemimethylated recognition sequence (Kd = 3.6 x 10(-7) M), but binds less well to unmethylated DNA (Kd = 8.3 x 10(-7) M). Surprisingly it shows specific, although poor, binding to fully methylated DNA (Kd = 4.2 x 10(-6) M). M.MspI binds approximately 5-fold more tightly to DNA containing its recognition sequence, CCGG, than to nonspecific sequences in the absence of cofactors. In the presence of S-adenosyl-L-methionine, S-adenosyl-L-homocysteine or sinefungin the discrimination between specific and non-specific sequences increases up to 100-fold. DNasel footprinting studies indicate that 16 base pairs of DNA are covered by M.MspI, with the recognition sequence CCGG located asymmetrically within the footprint.     

Initiation of the UvrABC nuclease cleavage reaction. Efficiency of incision is not correlated with UvrA binding affinity

The incision steps of Escherichia coli nucleotide excision repair are mediated by the UvrABC nuclease complex. We have previously shown that the UvrABC nuclease specifically incises apyrimidinic (AP) sites less efficiently than o-benzylhydroxylamine-modified apyrimidinic (BA) sites. To investigate these differences, quantitative DNase I footprinting titration studies were performed. The UvrA binding isotherms were similar for both the AP site (Kd = 6 x 10(-9) M) and the bulkier BA lesion (Kd = 14 x 10(-9) M), despite the fact that the extent of incision differs for these two lesions. It was also found that the relative binding affinity of the preincision UvrA2B complex to the AP and BA substrates differs significantly with estimated apparent equilibrium dissociation constants (Kd) of 4 x 10(-9) M and 80 x 10(-9) to 120 x 10(-9) M, respectively. These results indicate that incision efficiency does not correlate to UvrA binding affinity, but is a direct result of interactions between the UvrA2B complex and the site of the DNA damage. It is also shown that high UvrA concentrations are inhibitory to the UvrABC nuclease reaction.     

Interactions of norharman and harman with DNA.

The interactions of norharman (9H-pyrido [3,4-b] indole) and harman (1-methyl-9H-pyrido [3,4-b] indole) with DNA were studied. DNA caused remarkable fluorescence quenching and change in the absorption spectra of the dyes. Scatchard plots obtained by optical titration gave Kd values of 2.2 X 10(-5)M and 7.7 X 10(-6)M, and apparent numbers of binding sites of 0.13/base and 0.12/base for norharman and harman, respectively. Agarose gel electrophoresis of circular DNA, closed in the presence or absence of norharman revealed that the dye intercalates DNA, thereby causing 17 +/- 3 degrees unwinding of the double helix.     

苦瓜MAP30基因的毕赤酵母表达载体构建及重组菌株的筛选

目的:从苦瓜中克隆MAP30全长基因,并将该基因连接至表达载体pPIC9中,建立酵母菌落PCR筛选方法。方法采用改良SDS法从苦瓜表皮中提取基因组DNA,设计特异性的引物,通过PCR技术扩增出全长861bp的MAP30基因。该基因经XhoⅠ和EcoRⅠ双酶切,连接至毕赤酵母表达载体pPIC9中。重组载体转化GS115菌株,运用菌落PCR鉴定重组菌株。结果:基因测序表明,该基因已成功插入酵母表达载体pPIC9α-factor分泌信号下游,同源性分析表明该基因与GeneBank(AF284811)的核苷酸同源性达99.9%,氨基酸同源性达100%。菌落PCR显示外源基因已整合入酵母GS115菌株中。结论:成功地克隆了MAP30全长基因,并构建了含MAP30基因的重组毕赤酵母表达载体,并获得了整合菌株,为下一步研究奠定了基础。     

Shape-selective recognition of a model Okazaki fragment by geometrically-constrained bis-distamycins

Okazaki fragments represent interesting targets for the design of anticancer drugs because of their selective occurrence during DNA replication, a process often elevated in aggressive malignancies. Structural studies have indicated a bend occurs in the helical axis at the junction region (JR) that joins the DNA duplex region (DDR) and the RNA-DNA hybrid duplex region (HDR) of model Okazaki fragments. To identify a structural motif that provides a shape complementary to the Okazaki fragment minor groove, we have investigated the binding of geometrically-constrained bis-distamycins to a model Okazaki fragment, [OKA], with a sequence derived from the genome of simian virus 40 (SV40). Both the JR and the DDR of [OKA] contain consecutive A/T base pairs that could accommodate distamycin binding. Of the six bis-distamycins selected for analysis, the two with a para configuration of the distamycins on the benzene or pyridine scaffold bound [OKA] tightly (Kd approximately 10(-6) M from gel-shift assays; Kd approximately 10(-8) M from deltaT(M)) while the four with a meta orientation did not bind. The two mono-distamycins studied also did not bind [OKA]. Molecular modeling of the complex between the para bis-distamycin MT-9 and [OKA] revealed MT-9 adopted an S- shape complementary to the minor groove of the model Okazaki fragment.     

Oligomerization of Escherichia coli haemolysin (HlyA) is involved in pore formation

Summary The phycobilisome rod linker genes in the two closely related cyanobacteria Synechococcus sp. PCC 6301 and Synechococcus sp. PCC 7942 were studied. Southern blot analysis showed that the genetic organization of the phycobilisome rod operon is very similar in the two strains. The phycocyanin gene pair is duplicated and separated by a region of about 2.5 kb. The intervening region between the duplicated phycocyanin gene pair was cloned from Synechococcus sp. PCC 6301 and sequenced. Analysis of this DNA sequence revealed the presence of three open reading frames corresponding to 273, 289 and 81 amino acids, respectively. Insertion of a kanamycin resistance cassette into these open reading frames indicated that they corresponded to the genes encoding the 30, 33 and 9 kDa rod linkers, respectively, as judged by the loss of specific linkers from the phycobilisomes of the insertional mutants. Amino acid compositions of the 30 and 33 kDa linkers derived from the DNA sequence were found to deviate from those of purified 33 and 30 kDa linkers in the amounts of glutamic acid/glutamine residues. On the basis of similarity of the amino acid sequence of the rod linkers between Synechococcus sp. PCC 6301 and Calothrix sp. PCC 7601 we name the genes encoding the 30, 33 and 9 kDa linkers cpcH, cpcI and cpcD, respectively. The three linker genes were found to be co-transcribed on an mRNA of 3700 nucleotides. However, we also detected a smaller species of mRNA, of 3400 nucleotides, which would encode only the cpcH and cpcI genes. The 30 kDa linker was still found in phycobilisome rods lacking the 33 kDa linker and the 9 kDa linker was detected in mutants lacking the 33 or the 30 kDa linkers. Free phycocyanin was found in the mutants lacking the 33 or the 30 kDa linkers, whereas no free phycocyanin could be found in the mutant lacking the 9 kDa linker.Abbreviations PCC Pasteur Culture Collection - UTEX University of Texas Culture Collection The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank Nucleotide Sequence Databases under the accession number M94218     

Detection of ligation products of DNA linkers with 5'-OH ends by denaturing PAGE silver stain

To explore if DNA linkers with 5'-hydroxyl (OH) ends could be joined by commercial T4 and E. coli DNA ligase, these linkers were synthesized by using the solid-phase phosphoramidite method and joined by using commercial T4 and E. coli DNA ligases. The ligation products were detected by using denaturing PAGE silver stain and PCR method. About 0.5-1% of linkers A-B and E-F, and 0.13-0.5% of linkers C-D could be joined by T4 DNA ligases. About 0.25-0.77% of linkers A-B and E-F, and 0.06-0.39% of linkers C-D could be joined by E. coli DNA ligases. A 1-base deletion (-G) and a 5-base deletion (-GGAGC) could be found at the ligation junctions of the linkers. But about 80% of the ligation products purified with a PCR product purification kit did not contain these base deletions, meaning that some linkers had been correctly joined by T4 and E. coli DNA ligases. In addition, about 0.025-0.1% of oligo 11 could be phosphorylated by commercial T4 DNA ligase. The phosphorylation products could be increased when the phosphorylation reaction was extended from 1 hr to 2 hrs. We speculated that perhaps the linkers with 5'-OH ends could be joined by T4 or E. coli DNA ligase in 2 different manners: (i) about 0.025-0.1% of linkers could be phosphorylated by commercial T4 DNA ligase, and then these phosphorylated linkers could be joined to the 3'-OH ends of other linkers; and (ii) the linkers could delete one or more nucleotide(s) at their 5'-ends and thereby generated some 5'-phosphate ends, and then these 5'-phosphate ends could be joined to the 3'-OH ends of other linkers at a low efficiency. Our findings may probably indicate that some DNA nicks with 5'-OH ends can be joined by commercial T4 or E. coli DNA ligase even in the absence of PNK.     

A study of the binding of Mn2+ to bovine pancreatic deoxyribonuclease I and to deoxyribonucleic acid by electron paramagnetic resonance.

EPR studies of Mn2+ binding to bovine pancreatic deoxyribonuclease I show that the enzyme can bind three Mn2+ ions at pH 7.5 and 2 degrees. Two sites bind Mn2+ strongly, with a Kd of 10(-4)M, and the third binds Mn2+ weakly, with a Kd of 10(-3)M. Ca2+ competes with the two strong sites, whereas Mg2+ competes only with one of them, indicating that both sites are not equivalent. Mn2+ binding to DNA has been confirmed by EPR measurements. Two types of sites, with different affinities for Mn2+ binding, were found on DNA molecules, one with a Kd of 1.2 times 10(-4)M and the other with a Kd of 10(-3)M. Mg2+ ions can displace Mn2+ from the high affinity sites, but not from the low affinity sites. These results suggest the Mn2+ binds not only to the phosphate groups, but also to the electron donor groups of the base rings.     

Isolation and characterization of staphylocoagulase chymotryptic fragment. Localization of the procoagulant- and prothrombin-binding domain of this protein

Several strains of Staphylococcus aureus secrete a protein, staphylocoagulase, that binds stoichiometrically to human prothrombin, resulting in a coagulant complex designated staphylothrombin. In the present study, staphylocoagulase was digested with alpha-chymotrypsin and the resulting fragments were isolated by gel filtration. One fragment (Mr 43,000) exhibited a high affinity for human prothrombin (Kd = 1.7 X 10(-9) M), which is comparable to the affinity observed using intact staphylocoagulase (Kd = 4.6 X 10(-10) M). A complex of the Mr 43,000 fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. A second fragment (Mr 30,000) exhibited weaker affinity for prothrombin (Kd = 1.2 X 10(-7) M). While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. A third fragment (Mr 20,000) was found to bind to prothrombin, but the resultant complex did not exhibit clotting or amidase activity. Amino-terminal sequence analyses of these staphylocoagulase fragments revealed that the Mr 43,000 fragment constitutes the amino-terminal portion of staphylocoagulase and also contains the Mr 30,000 and 20,000 fragments. Moreover, the amino-terminal sequence of the Mr 20,000 fragment was identical to that observed for the Mr 30,000 fragment. From these results, we conclude that the functional region of staphylocoagulase for binding and activation of human prothrombin is localized in the amino-terminal region of the intact bacterial protein.     

Probing the primary structural determinants of streptokinase inter-domain linkers by site-specific substitution and deletion mutagenesis

The bacterial protein streptokinase (SK) contains three independently folded domains (α, β and γ), interconnected by two flexible linkers with noticeable sequence homology. To investigate their primary structure requirements, the linkers were swapped amongst themselves i.e. linker 1 (between α and β domains) was swapped with linker 2 (between β and γ domains) and vice versa. The resultant construct exhibited very low activity essentially due to an enhanced proteolytic susceptibility. However, a SK mutant with two linker 1 sequences, which was proteolytically as stable as WT-rSK retained about 10% of the plasminogen activator activity of rSK When the native sequence of each linker was substituted with 9 consecutive glycine sequences, in case of the linker 1 substitution mutant substantial activity was seen to survive, whereas the linker 2 mutant lost nearly all its activity. The optimal length of linkers was then studied through deletion mutagenesis experiments, which showed that deletion beyond three residues in either of the linkers resulted in virtually complete loss of activator activity. The effect of length of the linkers was then also examined by insertion of extraneous pentapeptide sequences having a propensity for adopting either an extended conformation or a relatively rigid conformation. The insertion of poly-Pro sequences into native linker 2 sequence caused up to 10-fold reduction in activity, whereas its effect in linker 1 was relatively minor. Interestingly, most of the linker mutants could form stable 1:1 complexes with human plasminogen. Taken together, these observations suggest that (i) the functioning of the inter-domain linkers of SK requires a critical minimal length, (ii) linker 1 is relatively more tolerant to insertions and sequence alterations, and appears to function primarily as a covalent connector between the α and β domains, and (iii) the native linker 2 sequence is virtually indispensable for the activity of SK probably because of structural and/or flexibility requirements in SK action during catalysis.     

Circular RNA oligonucleotides. Synthesis, nucleic acid binding properties, and a comparison with circular DNAs.

We report the synthesis and nucleic acid binding properties of two cyclic RNA oligonucleotides designed to bind single-stranded nucleic acids by pyr.pur.pyr-type triple helix formation. The circular RNAs are 34 nucleotides in size and were cyclized using a template-directed nonenzymatic ligation. To ensure isomeric 3'-5' purity in the ligation reaction, one nucleotide at the ligation site is a 2'-deoxyribose. One circle (1) is complementary to the sequence 5'-A12, and the second (2) is complementary to 5'-AAGAAAGAAAAG. Results of thermal denaturation experiments and mixing studies show that both circles bind complementary single-stranded DNA or RNA substrates by triple helix formation, in which two domains in a pyrimidine-rich circle sandwich a central purine-rich substrate. The affinities of these circles with their purine complements are much higher than the affinities of either the linear precursors or simple Watson-Crick DNA complements. For example, circle 1 binds rA12 (pH 7.0, 10 mM MgCl2, 100 mM NaCl) with a Tm of 48 degrees C and a Kd (37 degrees C) of 4.1 x 10(-9) M, while the linear precursor of the circle binds with a Tm of 34 degrees C and a Kd of 1.2 x 10(-6) M. The complexes of circle 2 are pH-dependent, as expected for triple helical complexes involving C(+)G.C triads, and mixing plots for both circles reveal one-to-one stoichiometry of binding either to RNA or DNA substrates. Comparison of circular RNAs with previously synthesized circular DNA oligonucleotides of the same sequence reveals similar behavior in the binding of DNA, but strikingly different behavior in the binding of RNA. The cyclic DNAs show high DNA-binding selectivity, giving relatively weaker duplex-type binding with complementary RNAs. The relative order of thermodynamic stability for the four types of triplex studied here is found to be DDD >> RRR > RDR >> DRD. The results are discussed in the context of recent reports of strong triplex dependence on RNA versus DNA backbones. Triplex-forming circular RNAs represent a novel and potentially useful strategy for high-affinity binding of RNA.     

Multiple defects of the nerve growth factor receptor in human neuroblastomas

Neuroblastoma is a tumor of postganglionic sympathetic origin, and nerve growth factor (NGF) is normally required for the survival and differentiation of sympathetic neuroblasts. Since the biological activity of NGF is mediated by the NGF receptor (NGFR), we hypothesized that defects in the NGF/NGFR pathway may play a role in maintenance of the undifferentiated state of neuroblastomas. To test this hypothesis, we examined the structure of the NGFR at the DNA, RNA, and protein levels in a panel of 10 neuroblastoma cell lines. In addition, we examined the function of the NGFR in these lines by analysis of NGF binding kinetics, as well as by the ability of NGF to induce c-fos expression and neurite outgrowth. Southern blot analysis showed that all 10 cell lines possess apparently normal NGFR genes. Northern blot and ligand binding/immunoprecipitation assays revealed four receptor-positive cell lines (NGP, NLF, SK-N-SH, and LA-N-6), with NGFR mRNA and protein of expected sizes (3.8 kilobases and Mr approximately 75,000, respectively). NGF binding assays and Scatchard analyses were performed on the four NGFR-positive lines. The NGP line possesses only low-affinity receptor (Kd approximately 3.5 x 10(-9)), whereas the other three lines express both low- and high-affinity forms (Kd approximately 10(-9) and Kd approximately 10(-11), respectively). However, none of the 10 lines exhibited a response to NGF treatment as assayed by c-fos mRNA induction and neurite extension.(ABSTRACT TRUNCATED AT 250 WORDS)     

High affinity interactions between the Alzheimer's beta-amyloid precursor proteins and the basement membrane form of heparan sulfate proteoglycan

High affinity interactions were studied between the basement membrane form of heparan sulfate proteoglycan (HSPG) and the 695-, 751-, and 770-amino acid Alzheimer amyloid precursor (AAP) proteins. Based on quantitative analyses of binding data, we identified single binding sites for the HSPG on AAP-695 (Kd = 9 x 10(-10) M), AAP-751 (Kd = 10 x 10(-9) M), and AAP-770 (Kd = 9 x 10(-9) M). It is postulated that the "Kunitz" protease inhibitor domain which is present in AAP-751 and -770 reduces the affinity of AAPs for the HSPG through steric hindrance and/or conformational alteration. HSPG binding was inhibited by heparin and dextran sulfate, but not by dermatan or chondroitin sulfate. HSPG protein core, obtained by heparitinase digestion, also bound to the beta-amyloid precursor proteins with high affinity, indicating that the high affinity binding site is constituted by the polypeptide chain rather than the carbohydrate moiety. The effects of various cations on these interactions were also studied. Our results suggest that specific interactions between the AAP proteins and the extracellular matrix may be involved in the nucleation stages of Alzheimer's disease type amyloidogenesis.     

Intermolecular association of the p185neu protein and EGF receptor modulates EGF receptor function

We have used cross-linking reagents on cell lines expressing both p185neu and EGFR. The lysates of the cells were precipitated with anti-p185neu or anti-EGFR antibodies. These precipitates included a high molecular weight complex that was identified as an EGFR-p185neu heterodimer. Heterodimerization was found to be induced by exposure to EGR. The EGFR of these cells displayed three affinity states for EGF: low (Kd, approximately 10(-9) M), high (Kd, 10(-9) to 10(-10) M), and very high (Kd, 10(-11) M), as determined by Scatchard analyses. Relatively small levels of EGF had a dramatic biological effect on cells expressing very high affinity EGFR. The very high affinity EGFR disappeared after the cells were treated with anti-p185neu monoclonal antibodies that selectively down-regulated p185neu. EGF and TPA had differential effects on down-modulation of the EGFR in cells that express either one or both species of receptor proteins.     

Psoralen photo-cross-linking by triplex-forming oligonucleotides at multiple sites in the human rhodopsin gene.

Targeting DNA damage by triplex-forming oligonucleotides (TFOs) represents a way of modifying gene expression and structure and a possible approach to gene therapy. We have determined that this approach can deliver damage with great specificity to sites in the human gene for the G-protein-linked receptor rhodopsin, mutations of which can lead to the genetic disorder autosomal dominant retinitis pigmentosa. We have introduced DNA monoadducts and interstrand cross-links at multiple target sites within the gene using TFOs with a photoactivatable psoralen group at the 5'-end. The extent of formation of photoadducts (i.e., monoadducts and cross-links) was measured at target sites with a 5'-ApT sequence at the triplex-duplex junction and at a target site with 5'-ApT and 5'-TpA sequences located four and seven nucleotides away, respectively. To improve psoralen reactivity at more distant sites, psoralen moieties were attached to TFOs with nucleotide "linkers" from two to nine nucleotides in length. High-affinity binding was maintained with linkers of up to 10 nucleotides, but affinities tended to decrease somewhat with increasing linker length due to faster dissociation kinetics. DNase I footprinting indicated little, if any, interaction between linkers and the duplex. Psoralen-TFO conjugates formed DNA cross-links with high efficiency (56-65%) at 5'-ApT sequences located at triplex junctions. At a 5'-ApT site four nucleotides away, the efficiency varied with linker length; a four-nucleotide linker gave the highest efficiency. Duplexes with 5'-TpA and 5'-ApT sites two nucleotides away, in otherwise identical sequences, were cross-linked with efficiencies of 56 and 38%, respectively. These results indicate that TFO-linker-psoralen conjugates allow simultaneous, efficient targeting of multiple sites in the human rhodopsin gene.