Electron microscopic studies of sequence-specific recognition of duplex DNA by different ligands

The following ligands were used to study sequence specific recognition of duplex DNA by electron microscopic techniques: methyltransferases BspR1 and EcoR124 (recognition sequences GGCC and GAAN7RTCG, respectively), a biotinylated deoxyoligonucleotide 5′-CTCTCTCTCTCTCT-3′ capable of forming triplex DNA, and PNA oligomer H-T₁₀-LysNH₂. For each ligand the best conditions for electron microscopic (EM)detection of stable specific complex formation were determined. It was demonstrated that EM allowed us to determine the position of the individual target site with an error of 15–20 bp, the relative affinities for individual target sites and kinetic parameters of the binding. These results open new possibilities for EM investigations of sequence-specific interactions with a wide range of other ligands of a similar nature. They also imply that a wide range of different sequences can be unambiguously and precisely mapped by EM and greatly extend the scope of EM applications for physical mapping of genomic DNA.     

Resistance of bacteriophage H1 to restriction and modification by Bacillus subtilis R

H1, a 5-hydroxymethyluracil (HMU)-containing Bacillus subtilis bacteriophage, was neither restricted nor modified upon infection of B. subtilis R cells. In vitro, H1 DNA was not restricted by BsuR under standard conditions (200 mM salt), although the expected frequency of -GGCC- cleavage sites was approximately 250. However, four specific sites were cleaved under nonstandard conditions (low salt or high pH) or in the presence of organic solvents, like dimethyl sulfoxide and glycerol. After the substitution of thymine for HMU by DNA cloning in B. subtilis, a BsuR cleavage site was restricted and modified under standard conditions. No additional sites were detected after shotgun-cloning of about 11% of the chromosome. The nucleotide sequence of a cleavage site was found to be 5'. .C-A-Hmu-A-A-C-Hmu-Hmu-Hmu-G-G-C-C-Hmu-A-G-. . .3', which shows the presence of a bona fide BsuR (GGCC) recognition sequence, flanked by (Hmu-A)-rich sequences. The results suggested that the resistance of H1 to restriction and modification by B. subtilis R was due to (i) a strong bias against the GGCC-recognition sequence and (ii) protection of the four remaining GGCC sites as a consequence of HMU-A base pairs flanking the sites.     

Sequence based high resolution chromosomal CGH

We aimed to directly align a chromosomal CGH (cCGH) pattern with the gene mapping data by taking advantage of the clustering of the GGCC motif at certain positions in the human genome. The alignment of chromosomal with sequence data was achieved by superimposition of (i) the fluorescence intensity of the sequence specific fluorochrome, Chromomycin A3 (CMA3), (ii) the cCGH fluorescence intensity profile of individual chromosomes and (iii) the GGCC density profile extracted from the Ensembl genome sequence database. The superimposition of these three pieces of information allowed us to precisely localize regions of amplification in the neuroblastoma cell line STA-NB-15. Two prominent cCGH peaks were noted, one at 2p24.3, the position 15.4 mega base (Mb), and the other at 2p23.2, 29.51 Mb. FISH and high resolution array CGH (aCGH) experiments disclosed an amplification of MYCN (16 Mb) and ALK (29.2-29.9 Mb), thus confirming the cCGH data. The combined visualization of sequence information and cCGH data drastically improves the resolution of the method to less than 2 Mb.     

Cytosine methylation by the SuaI restriction-modification system: implications for genetic fidelity in a hyperthermophilic archaeon

5-methylcytosine in chromosomal DNA represents a potential source of frequent spontaneous mutation for hyperthermophiles. To determine the relevance of this threat for the archaeon Sulfolobus acidocaldarius, the mode of GGCC methylation by its restriction-modification system, SuaI, was investigated. Distinct isoschizomers of the SuaI endonuclease were used to probe the methylation state of GGCC in native S. acidocaldarius DNA. In addition, the methylation sensitivity of the SuaI endonuclease was determined with synthetic oligonucleotide substrates and modified natural DNAs. The results show that the SuaI system uses N(4) methylation to block cleavage of its recognition site, thereby avoiding the creation of G. T mismatches by spontaneous deamination at extremely high temperature.     

Restriction and modification in Bacillus subtilis: nucleotide sequence, functional organization and product of the DNA methyltransferase gene of bacteriophage SPR

Bacillus subtilis phage SPR codes for a DNA methyltransferase (Mtase) which methylates the 5' cytosine in the sequence GGCC and both cytosines in the sequence CCGG. A 2126-bp fragment of SPR DNA containing the Mtase gene has been sequenced. This fragment has only one significant open reading frame of 1347 bp, which corresponds to the Mtase gene. Within the sequence the Mtase promoter has been defined by S1 mapping. The size of the SPR Mtase predicted from the deduced amino acid composition is 49.9 kDal. This is in agreement with both the Mr of the purified enzyme and with that of the SPR Mtase gene product identified here by minicell technique. Base changes leading to mutants affected in Mtase activity were localized within the Mtase gene.     

Electron microscopic mapping of proteins bound to herpes simplex virus DNA.

Exonuclease digestion experiments have suggested that there is a protein(s) bound close to one or both ends of herpes simplex virus-1 (HSV) DNA. The existence of such bound proteins has been positively demonstrated and their positions on the HSV genome determined by application of a newly developed method for electron microscopic mapping of proteins bound to nucleic acids. Purified HSV DNA was treated with dinitrofluorobenzene under conditions that covalently attach the dinitrophenyl (DNP) group to the proteins in a protein-nucleic acid complex. The HSV DNA-protein-(DNP)n complex was treated with rabbit anti-DNP IgG, and, in some cases, additionally treated with monovalent Fab fragments of goat anti-rabbit IgG, and mounted for examination in the electron microscope. Electron opaque dots representing the protein-(DNP)n-(IgG)m complex were seen on the HSV DNA. Direct measurements of the positions of the protein, as well as partial denaturation mapping, indicate that there are four positions for protein bound to HSV DNA: two near but not at the two ends and two at sites corresponding to the internal inverted repeats of the ends. These results suggest that there is a specific protein binding sequence within the direct terminal repeat of HSV DNA. The previous observation that HSV DNA is more sensitive to digestion by a 3' than by a 5' exonuclease then indicates that the bound protein(s) is more intimately associated with one strand of the specific sequence than with the complementary strand.     

Bacillus subtilis phage SPR codes for a DNA methyltransferase with triple sequence specificity.

SPR, a temperate Bacillus subtilis phage, codes for a DNA methyltransferase that can methylate the sequences GGCC (or GGCC) and CCGG at the cytosines indicated. We show here that it can also methylate the sequence CC(A/T)GG and protect it from cleavage with EcoRII and ApyI. This methylation can be seen in vivo as well as in vitro with purified SPR methyltransferase. SPR19 and SPR83 are two mutant phages, defective in GGCC or CCGG methylation, respectively. These mutants have not lost their ability to methylate CC(A/T)GG sites. Mutation SPR26 has lost the ability to methylate all three sites. Thus the SPR methyltransferase codes for three genetically distinguishable methylation abilities.     

Interaction between the cyclic AMP receptor protein and DNA. Conformational studies

The binding of the cyclic adenosine 3',5' monophosphate receptor protein (CRP or CAP) of Escherichia coli to non-specific DNA and to a specific lac recognition sequence has been investigated by circular dichroism (c.d.) spectroscopy. The effect of cAMP and cGMP on the co-operative non-specific binding was also studied. For the non-specific binding in the absence of cAMP a c.d. change (decrease of the intensity of the positive band with a shift of its maximum to longer wavelength) indicates that the DNA undergoes a conformational change upon CRP binding. This change might reflect the formation of the solenoidal coil previously observed by electron microscopy. The amplitude of the c.d. change increases linearly with the degree of saturation of the DNA and does not depend on the size of the clusters of CRP bound. From the variation of the c.d. effect as a function of the ionic strength, the product K omega (K, the intrinsic binding constant and omega, the co-operativity parameter) could be determined. The number of ion pairs involved in complex formation between CRP and DNA was found to be six to seven. Experiments performed with several DNAs, including the alternating polymers poly[d(A-T)] and poly[d(G-C)], demonstrated that the conformational change does not depend on the DNA sequence. However, in the presence of cAMP the c.d. spectrum of the DNA shows only a small variation upon binding CRP. In contrast, in the presence of cGMP the conformational change of the DNA is similar to that observed when non-liganded CRP binds. For the specific lac operon binding, the c.d. change is different from those observed for non-specific binding in the presence or absence of cAMP. These results emphasize the high variability of the DNA structure upon binding the same protein.     

灵杆菌非特异性核酸酶的原核表达、纯化及活性分析

以灵杆菌基因组DNA为模板,PCR扩增非特异性核酸酶 (Non-specific nuclease,NU) 基因,并克隆到pMAL-c4X载体上构建重组表达载体pMAL-c4X-NU。经测序及 BLASTN发现其与灵杆菌Serratia marcescens核酸酶基因的同源性为97％。将构建的表达载体pMAL-c4X-NU转入大肠杆菌BL21,经IPTG诱导实现了胞内表达78 kDa的麦芽糖结合蛋白-NU融合蛋白 (Maltose-binding protein-NU,MBP-NU),其最佳诱导表达条件为37 ℃,0.75 mmol/L IPTG诱导1.5 h。用Amylose resin纯化得到了目的蛋白。活性检测表明MBP-NU具有同时降解DNA和RNA的活性,在37 ℃、pH 8.0时活性最高,比活力为1.11×106 U/mg,目标蛋白的纯化效率可达10.875 mg/L。纯化的目标蛋白中无蛋白酶活性存在。0.5 mmol/L乙二胺四乙酸 (Ethylene diamine tetraacetic acid,EDTA)、1 mmol/L苯甲基磺酰氟 (Phenylmethanesulfonyl fluoride,PMSF) 以及150 mmol/L KCl对MBP-NU的活性几乎无影响,因此MBP-NU可作为蛋白质纯化过程中核酸的高效降解酶。     

Structures during binding of cAMP receptor to promoter DNA: promoter search slowed by non-specific sites

The kinetics of cAMP receptor (CAP) binding to promoter DNA has been studied by stopped-flow electric-dichroism at a reduced salt concentration, where the coupling of non-specific and specific binding can be observed directly. Amplitudes, rise and decay times of dichroism transients provide detailed information about the reaction and the structure of intermediates over more than six orders of magnitude on the time scale. CAP binding during the first milliseconds after mixing is indicated by an increase of both rise- and decay-time constants. A particularly large increase of rise times reflects initial formation of non-symmetric complexes by protein binding to non-specific sites at DNA ends. The increase of the hydrodynamic dimensions continues up to ~1 s, before a decrease of time constants reflects transition to compact states with bent DNA up to the time range of ~10³ s. The slow approach to CAP-induced DNA bending is due to non-specific complexes, which are formed initially and are converted slowly to the specific complex. At the salt concentration of 13.5 mM, conversion to specific complexes with bent DNA is completed after ~40 s at pH 8 compared to >10³ s at pH 7, resulting from a higher affinity of CAP to non-specific sites at pH 7 than 8 by a factor of ~100. Thus, under the given conditions non-specific sites delay rather than facilitate formation of the specific complex with bent DNA. Experimental data obtained for a non-specific DNA clearly indicate the impact of pseudo-sites. The different electro-optical parameters have been combined in global fits.     

CAP binding to B and Z forms of DNA.

We have examined the interaction between the cyclic AMP receptor protein (CAP) and a small DNA fragment containing its specific recognition sequence by circular dichroism spectroscopy. The binding of CAP to this fragment induces a B to "C-like" change in the CD spectrum, which is different from that observed for non-specific binding. A one-to-one (CAP dimer to DNA) binding stoichiometry was deduced from spectroscopic titration data, as was a non-specific binding site size of 17 bp/dimer. In addition, we have compared the non-specific binding affinity of CAP for the B and Z forms of synthetic DNA copolymers. A slight preference for the B form was found. These results do not support the recent specific suggestion that CAP binds to a left-handed form of DNA (1), but indicate more generally that an optically detectable conformational change takes place in DNA on binding CAP.     

Full-length cDNA cloning utilizing the polymerase chain reaction, a degenerate oligonucleotide sequence and a universal mRNA primer.

A full-length cDNA was selectively amplified by the polymerase chain reaction (PCR) utilizing a primer pair consisting of a "universal" 21-base synthetic deoxyoligonucleotide (oligo dT 17GGCC) and a specific degenerate deoxyoligonucleotide sequence (DOS) derived from the N-terminal amino acid sequence. This double-stranded amplified cDNA was uni-directionally cloned into M13mp19 utilizing two restriction sites that had been previously incorporated into the termini of the universal and specific DOS primers. Cloning of the specific cDNA via this PCR amplification with the universal/specific DOS primer pair approach was confirmed by screening with a second DOS contiguous with the DOS employed to prime second (sense)-strand cDNA synthesis. This technique allows for the selective full-length cDNA cloning of low-abundance mRNAs from a single-protein sequence determination.     

Specifically unmethylated cytidylic-guanylate sites in Herpesvirus saimiri DNA in tumor cells.

The restriction endonucleases MspI (CCGG), HpaII (CCGG), FnuDII (CGCG), and HaeIII (GGCC) were used to study the methylation of Herpesvirus saimiri DNA in tumor cells taken directly from tumor-bearing animals. No evidence was found for methylation of the 5' terminal C in the sequence CCGG or of the internal C in the sequence GGCC, but extensive methylation of CG was detected. Fifteen HpaII sites and 17 FnuDII sites were detected in the unique DNA region of the H. saimiri strain used. Twenty-eight of the 32 sites were methylated in greater than 90% of the viral DNA molecules in tumor cells, but the remaining 4 sites were unmethylated in greater than 95% of the viral DNA molecules in tumor cells. The locations of the four specifically unmethylated sites were mapped and appeared to be identical in the four different induced leukemias examined (one owl monkey and three white-lipped marmosets). The nonproducer 1670 tumor cell line, in continuous passage for over 7 years, contained four similar specifically unmethylated sites. Possibilities for the physiological significance of the unmethylated sites are discussed.     

Sequential order of target-recognizing domains in multispecific DNA-methyltransferases.

In the multispecific DNA(cytosine-5)-methyltransferases (Mtases) of Bacillus subtilis phages SPR and phi 3T the domains responsible for recognition of DNA methylation targets CCA/TGG, CCGG, GGCC (SPR) and GCNGC, GGCC (phi 3T) represent contiguous sequences of approximately 50 amino acids each. These domains are tandemly arranged and do not overlap. They are part of a 'variable' segment within the enzymes which is flanked by 'conserved' amino acids, which are very similar amongst bacterial monospecific and the multispecific Mtases studied here. These results follow from a mutational analysis of the SPR and phi 3T Mtase genes. They further support our concept of a modular enzyme organization, according to which variability of type II Mtases with respect to target recognition is achieved by a combination of the same enzyme core with a variety of target-recognizing domains.     

Mapping subunit location on the Saccharomyces cerevisiae origin recognition complex free and bound to DNA using a novel nanoscale biopointer

The Saccharomyces cerevisiae origin recognition complex (ORC) is composed of six subunits and is an essential component in the assembly of the replication apparatus. To probe the organization of this multiprotein complex by electron microscopy, each subunit was tagged on either its C or N terminus with biotin and assembled into a complex with the five other unmodified subunits. A nanoscale biopointer consisting of a short DNA duplex with streptavidin at one end was used to map the location of the N and C termini of each subunit. These observations were made using ORC free in solution and bound to the ARS1 origin of replication. This mapping confirms and extends previous studies mapping the sites of subunit interaction with origin DNA. In particular, we provide new information concerning the stoichiometry of the ORC-ARS1 complex and the changes in conformation that are associated with DNA binding by ORC. This versatile, new approach to mapping protein structure has potential for many applications.     

Theoretical analysis of ''addressed'' chemical modification of DNA.

Chemical "addressed" modification of DNA involves treatment of single-stranded DNA with oligonucleotides complementary to certain target sequences in this DNA and bearing a groupings reactive towards DNA bases. The binding of oligonucleotides can occur both at completely (specific) and incompletely (nonspecific) complementary sites. We analyse the modification of a fragment that is flanked by two target sequences complementary to a given oligonucleotide address, contains no more such targets and has some randomly distributed sites for nonspecific binding. Conditions for the maximum ratio between specific and non-specific modification are determined. We find the probability of both target termini being specifically modified without any non-specific modification occurring within the fragment up to a given moment in time. Quantitative analysis is based on the use of known features of the specific and non-specific binding of an oligonucleotide to DNA sites. This analysis shows the possibility of specific cutting of DNA based on addressed modification.