Cytosine methylation enhances Z-DNA formation in vivo.

The influence of cytosine methylation on the supercoil-stabilized B-Z equilibrium in Escherichia coli was analyzed by two independent assays. Both the M.EcoRI inhibition assay and the linking-number assay have been used previously to establish that dC-dG segments of sufficient lengths can exist as left-handed helices in vivo. A series of dC-dG plasmid inserts with Z-form potential, ranging in length from 14 to 74 base pairs, was investigated. Complete methylation of cytosine at all HhaI sites, including the inserts, was obtained by coexpression of the HhaI methyltransferase (M.HhaI) in cells also carrying a dC-dG-containing plasmid. Both assays showed that for all lengths of dC-dG inserts, the relative amounts of B and Z helices were shifted to more Z-DNA in the presence of M.HhaI than in the absence of M.HhaI. These results indicate that cytosine methylation enhances the formation of Z-DNA helices at the superhelix density present in E. coli. The B-Z equilibrium, in combination with site-specific base methylation, may constitute a concerted mechanism for the modulation of DNA topology and DNA-protein interactions.     

Tetracycline promoter mutations decrease non-B DNA structural transitions, negative linking differences and deletions in recombinant plasmids in Escherichia coli

The ability to clone a variety of sequences with varying capabilities of adopting non-B structures (left-handed Z-DNA, cruciforms or triplexes) into three loci of pBR322 was investigated. In general, the inserts were stable (non-deleted) in the EcoRI site (an untranslated region) of pBR322. However, sequences most likely to adopt left-handed Z-DNA or triplexes in vivo suffered deletions when cloned into the BamHI site, which is located in the tetracycline resistance structural gene (tet). Conversely, when the promoter for the tet gene was altered by filling-in the unique HindIII or ClaI sites, the inserts in the BamHI site were not deleted. Concomitantly, the negative linking differences of the plasmids were reduced. Also, inserts with a high potential to adopt Z-DNA conformations were substantially deleted in the PvuII site of pBR322 (near the replication origin and the copy number control region), but were less deleted if the tet promoter was insertion-mutated. The deletion phenomena are due to the capacity of these sequences to adopt left-handed Z-DNA or triplexes in vivo since shorter inserts, less prone to form non-B DNA structures, or random sequences, did not exhibit this behavior. Sequences with the potential to adopt cruciforms were stable in all sites under all conditions. These results reveal a complex interrelationship between insert deletions (apparently the result of genetic recombination), negative supercoiling, and the formation of non-B DNA structures in living Escherichia coli cells.     

Isolation of Bacillus subtilis genes from a charon 4A library.

A library of Bacillus subtilis chromosomal deoxyribonucleic acid (DNA) was constructed, using lambda charon 4A as a cloning vector. Partially cleaved Bacillus subtilis DNA was prepared by partial methylation with EcoRI methylase, followed by complete EcoRI endonuclease digestion. More than 95% of the phage particles carried B. subtilis DNA inserts. When this library was screened for transforming activity, using competent cells, 70% of the genetic markers tested were found in a sample of 1,710 plaques. Cloned genetic loci were found to be about 100-fold more efficient in transforming activity than chromosomal DNA. Intact phage particles containing the pheA locus were found to be able to transform competent recipients with approximately the same efficiency as phage DNA. Transformation by intact particles was insensitive to deoxyribonuclease.     

Transfection of molecularly cloned Friend murine leukemia virus DNA yields a highly leukemogenic helper-independent type C virus.

Unintegrated viral DNA was isolated via the Hirt procedure from mouse fibroblasts newly infected with Friend murine leukemia virus (F-MuLV) clone 201, a biologically cloned helper virus isolated from stocks of F-MuLV complex. A physical map of the unintegrated in vivo linear viral DNA was generated for several restriction endonucleases. The supercoiled viral DNA was digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized viral DNA was then inserted into lambda gtWES.lambda B at the EcoRI site and cloned in an approved EK2 host. Eight independent lambda-mouse recombinants were identified as containing F-MuLV DNA inserts by hybridization with F-MuLV 32P-labeled complementary DNA. One of the F-MuLV DNA inserts was 9.1 kilobases (kb) and had the same restriction enzyme sites as the unintegrated linear F-MuLV DNA. Six inserts were 8.5 kb; each lacked a single copy of the terminally redundant sequences of the unintegrated linear viral DNA. One insert was 8.2 kb and contained a 0.9-kb deletion. After digestion with EcoRI, one recombinant DNA preparation containing an 8.5-kb insert was infectious for NIH 3T3 cells. Undigested recombinant DNA was not infectious. The infectivity of the EcoRI-digested DNA followed multihit kinetics, indicating that more than one molecule was required to register as an infectious unit. The virus isolated from this transfection (F-MuLV-57) was NB-ecotropic, helper-independent, and formed XC plaques. Inoculation of this virus into newborn NIH Swiss mice induced leukemia and splenomegaly in greater than 90% of animals within 3 to 4 weeks. The gross and microscopic abnormalities induced by F-MuLV clone 57 were identical to those seen with the original parent stocks of F-MuLV clone 201. These results indicate that this helper-independent F-MuLV can induce a rapid nonthymic leukemia in the absence of the spleen focus-forming virus.     

Intervening sequences in human fetal globin genes adopt left-handed Z helices

The large intervening sequences ( IVS2 ) of three human fetal globin genes contain tracts of alternating purine-pyrimidine sequences approximately 40-60 base pairs in length which adopt left-handed Z DNA helices under the influence of negative supercoiling. The amount of negative supercoiling (approximately 0.045) required for the right- to left-handed transitions is within the physiological range. The structural aberrations between the right- and left-handed helices were mapped by sequencing the S1 nuclease cleavage sites. Two-dimensional gel electrophoretic analyses of the supercoil-induced relaxation served to characterize the type and length of left-handed structure. Furthermore, binding studies with several types of antibodies confirmed the presence of left-handed helices. Since these simple sequences appear to be hotspots for recombination and gene conversion, unusual DNA conformations may participate in genetic expression.     

Three-dimensional structure of complexes of single-stranded DNA-binding proteins with DNA. IKe and fd gene 5 proteins form left-handed helices with single-stranded DNA

Specimen-tilting in an electron microscope was used to determine the three-dimensional architecture of the helical complexes formed with DNA by the closely related single-stranded DNA binding proteins of fd and IKe filamentous viruses. The fd gene 5 protein is the only member of the DNA-helix-destabilizing class of proteins whose structure has been determined crystallographically, and yet a parameter essential to molecular modeling of the co-operative interaction of this protein with DNA, the helix handedness, has not been available prior to this work. We find that complexes formed by titrating fd viral DNA with either the fd or IKe gene 5 protein have a left-handed helical sense. Complexes isolated from Escherichia coli infected by fd virus are also found to be left-handed helical; hence, the left-handed fd helices are not an artefact of reconstitution in vitro. Because the proteins and nucleic acid of the complexes are composed of asymmetric units which cannot be fitted equivalently to right-handed and left-handed helices, these results rule out a previous computer graphics atomic model for the helical fd complexes: a right-handed helix had been assumed for the model. Our work provides a defined three-dimensional structural framework within which to model the protein-DNA and protein-protein interactions of two structurally related proteins that bind contiguously and co-operatively on single-stranded DNAs.     

In vivo specificity of EcoRI DNA methyltransferase.

The EcoRI adenine DNA methyltransferase forms part of a bacterial restriction/modification system; the methyltransferase modifies the second adenine within the canonical site GAATTC, thereby preventing the EcoRI endonuclease from cleaving this site. We show that five noncanonical EcoRI sites (TAATTC, CAATTC, GTATTC, GGATTC and GAGTTC) are not methylated in vivo under conditions when the canonical site is methylated. Only when the methyltransferase is overexpressed is partial in vivo methylation of the five sites detected. Our results suggest that the methyltransferase does not protect host DNA against potential endonuclease-mediated cleavage at noncanonical sites. Our related in vitro analysis of the methyltransferase reveals a low level of sequence-discrimination. We propose that the high in vivo specificity may be due to the active removal of methylated sequences by DNA repair enzymes (J. Bacteriology (1987), 169 3243-3250).     

Methylation of cytosine in the 5-position alters the structural and energetic properties of the supercoil-induced Z-helix and of B-Z junctions

The structural and energetic consequences of cytosine methylation in the 5-position on the supercoil-dependent B-Z equilibrium in alternating dC-dG sequences cloned into recombinant plasmids were investigated. The helical parameters determined with the band shift method for right-handed [10.7 base pairs (bp)/turn] and left-handed (12.8 bp/turn) 5MedC-dG inserts were different from the helical repeat values for unmethylated dC-dG inserts (10.5 bp/turn in the right-handed and 11.5 bp/turn in the left-handed form). We analyzed the thermodynamic parameters delta GBZ (free energy difference per base pair between right-handed and left-handed helix structure), delta Gjx (free energy for formation of one B-Z junction), and b (helix unwinding at a junction region) for varying lengths of dC-dG inserts by two-dimensional gel electrophoresis and application of a statistical mechanics model. A comparison of plasmids fully methylated in vitro with HhaI methylase and their unmethylated counterparts revealed that delta Gjx is not significantly changed by cytosine methylation. However, this base modification results in an approximate 3-fold decrease of delta GBZ and an approximate 2-fold decrease of the unwinding b at B-Z junction regions. Analysis of a pair of related plasmids, each containing two dC-dG blocks, revealed qualitatively different transition behaviors. When the two dC-dG blocks were separated by 95 bp of a mixed sequence, they underwent independent B to Z transitions with separate nucleation events and junction formations. When the two blocks were separated by only a 4 bp GATC sequence, only one nucleation event was necessary, and the Z-helix spread across the nonalternating GATC region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic heterogeneity of steroid sulfatase deficiency revealed with cDNA for human steroid sulfatase

Three cDNA clones with inserts of 1.2-1.6 kb that reacted both with antibodies and oligonucleotides specific for steroid sulfatase were isolated from a human placental library in lambda gt11. The 5'-end of one of the inserts, STS-3, was sequenced and colinearity with the amino acid sequence of 3 peptides of steroid sulfatase encompassing 64 amino acids was demonstrated. STS-3 hybridized with 2.5, 4.6 and 6.3 kb species in poly(A)+RNA and with 2.5, 4 and 9 kb fragments of EcoRI digested human DNA. The frequency of the EcoRI fragments in DNA from females was approximately twice that in DNA from males. DNA from two patients with steroid sulfatase deficiency and X-linked ichthyosis did not hybridize with STS-3. DNA from a third patient showed a normal hybridization pattern. It is concluded that steroid sulfatase deficiency is a genetically heterogenous disorder.     

Demethylation of satellite I DNA during senescence of bovine adrenocortical cells in culture.

Over the finite proliferative life span of cultured bovine adrenocortical cells, satellite I DNA shows a progressive and extensive loss of methylation at CCGG sites. This was shown by Southern blotting after digestion with the methylation-sensitive enzyme HpaII alone, which provides a sensitive indicator of methylation loss, or digestion with the combination of EcoRI and HpaII, which provides a quantitative indication of loss of methylation. Bovine tissues, including adrenal cortex, all showed a much higher level of satellite methylation than cultured adrenocortical cells. After adrenocortical cells are placed in culture, some demethylation of satellite I is seen as early as 10 population doublings. By 80 population doublings, loss of satellite DNA methylation is extensive. The loss does not appear to prevent continued cell division, since an extended life span clone of bovine adrenocortical cells transfected with SV40 T antigen showed a similar pattern of extensive demethylation. Satellite demethylation has been reported in aging in vivo and the present cell culture system may provide an in vitro model for this form of genetic instability.     

Potassium permanganate as an in situ probe for B-Z and Z-Z junctions.

The availability of DNA structural probes that can be applied to living cells is essential for the analysis of biological functions of unusual DNA structures adopted in vivo. We have developed a chemical probe assay to detect and quantitate left-handed Z-DNA structures in recombinant plasmids in growing E. coli cells. Potassium permanganate selectively reacts with B-Z or Z-Z junction regions in supercoiled plasmids harbored in the cells. Restriction enzyme recognition sites located at these junctions are not cleaved by the corresponding endonuclease after modification with KMnO4. This inhibition of cleavage allows the determination of the relative amounts of B- and Z-forms of the cloned inserts inside the cell. We have successfully applied this method to monitor the extent of Z-DNA formation in E. coli as a function of the growth phase and mutated topoisomerase or gyrase activities. The assay can in principle be used for any unusual DNA structure that contains a restriction recognition site inside or near the structural alteration. It can be a useful tool to analyze in vivo correlations between DNA structure and gene regulatory events.     

Construction of an ordered overlapping library of bacteriophage P1 DNA in phage vector lambda D69

A library of bacteriophage P1 DNA was constructed in the phage vector lambda D69. The DNA of some 150 randomly chosen lambda-P1 hybrid phages containing P1 DNA fragments 5-10 kb in size was analyzed by restriction endonuclease digestion using enzymes EcoRI, BglII, and BamHI that cleave P1 DNA at known positions on the physical map of P1. Approximately one third of the phages contained P1 DNA inserts having two or more restriction sites for any one of these enzymes, thus allowing the location of the insert to be determined with respect to the physical map. Genetic tests allowed detection of lambda-P1 hybrid phages possessing inserts with functional P1 ban and CmR genes. A subset of 18 phages was analyzed in more detail; their P1 DNA inserts comprise an ordered collection of overlapping P1 DNA fragments that cover almost 98% of the P1 genome.     

Multiple DNA secondary structures in perfect inverted repeat inserts in plasmids. Right-handed B-DNA, cruciforms, and left-handed Z-DNA

The capabilities of five recombinant plasmids, containing relatively long (approximately 60-100 base pairs) perfect inverted repeat (IR) inserts, to support supercoil stabilized non-B-DNA structures were studied in vitro. The IRs were also alternating purine-pyrimidine sequences, thus, each could form either left-handed Z-DNA or cruciforms. Single-strand specific endonucleases, restriction endonucleases and methylases, and OsO4 modifications were used to characterize the DNA structures. Two-dimensional gel electrophoretic studies indicated that three of the five IRs formed both cruciforms and Z-DNA. (C-G) containing inserts preferred to form Z-DNA, whereas (T-G) sequences favored cruciforms. In vivo supercoil relaxation experiments demonstrated the existence of cruciforms in Escherichia coli. The physiological significance of these structures is discussed.     

Melon bacterial artificial chromosome (BAC) library construction using improved methods and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2).

Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that co-segregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.     

Molecular cloning of the Harvey sarcoma virus closed circular DNA intermediates: initial structural and biological characterization.

Supercoiled Harvey sarcoma virus (Ha-SV) DNA was extracted from newly infected cells by the Hirt procedure, enriched by preparative agarose gel electrophoresis, and digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized Ha-SV DNA was then inserted into lambda gtWESlambda B at the EcoRI site and cloned in an approved EK2 host. Ha-SV DNA inserts from six independently derived recombinant clones have been analyzed by restriction endonuclease digestion, molecular hybridization, electron microscopy, and infectivity. Four of the Ha-SV DNA inserts were identical, contained about 6.0 kilobase pairs (kbp), and comigrated in agarose gels with the infectious, unintegrated, linear Ha-SV DNA. One insert was approximately 0.65 kbp smaller (5.35 kbp) and one was approximately 0.65 kpb larger (6.65 kpb) than the 6.0 kpb inserts. R-looping with Ha-SV RNA revealed that the small (5.35 kbp) insert contained one copy of the Ha-SV RNA. Preliminary restriction endonuclease digestion of the recombinant DNAs suggested that the middle-size inserts contained a 0.65-kbp tandem duplication of sequences present only one in the small-size insert; this duplication corresponded to the 0.65-kpb terminal duplication of the unintegrated linear Ha-SV DNA. The large-size insert apparently contained a tandem triplication of these terminally located sequences. DNA of all three sized inserts induced foci in NIH 3T3 cells, and focus-forming activity could be rescued from the transformed cells by superinfection with helper virus. Infectivity followed single-hit kinetics, suggesting that the foci were induced by a single molecule.     

Methylation of intact chromosomes by bacterial methylases in agarose plugs suitable for pulsed-field electrophoresis. Methylation of intact chromosomes in agarose by methylases

Conditions were determined for the methylation of intact yeast chromosomes by EcoRI, HhaI, and MspI bacterial methylases using an endonuclease protection assay while the chromosomes were embedded in agarose plugs suitable for transverse-field electrophoresis. Parameters were also established for the methylation of human chromosomes by EcoRI methylase. Methylation of embedded chromosomes by EcoRI methylase required prewashes with EDTA. EcoRI, HhaI, and MspI methylases showed optimal activity when nonacetylated bovine serum albumin, high levels of S-adenosylmethionine, and high levels of methylase were used. The use of bacterial methylases for methylation of embedded chromosomes will allow investigators to normalize variations in cellular DNA methylation prior to restriction and create new and rare endonuclease recognition sites which will facilitate the detection of chromosomal alterations and deletions.     

Differential stability of DNA crossovers in solution mediated by divalent cations

The assembly of DNA duplexes into higher-order structures plays a major role in many vital cellular functions such as recombination, chromatin packaging and gene regulation. However, little is currently known about the molecular structure and stability of direct DNA–DNA interactions that are required for such functions. In nature, DNA helices minimize electrostatic repulsion between double helices in several ways. Within crystals, B-DNA forms either right-handed crossovers by groove–backbone interaction or left-handed crossovers by groove–groove juxtaposition. We evaluated the stability of such crossovers at various ionic concentrations using large-scale atomistic molecular dynamics simulations. Our results show that right-handed DNA crossovers are thermodynamically stable in solution in the presence of divalent cations. Attractive forces at short-range stabilize such crossover structures with inter-axial separation of helices less than 20 Å. Right-handed crossovers, however, dissociate swiftly in the presence of monovalent ions only. Surprisingly, left-handed crossovers, assembled by sequence-independent juxtaposition of the helices, appear unstable even at the highest concentration of Mg²⁺studied here. Our study provides new molecular insights into chiral association of DNA duplexes and highlights the unique role divalent cations play in differential stabilization of crossover structures. These results may serve as a rational basis to understand the role DNA crossovers play in biological processes.     

Bending and flexibility of methylated and unmethylated EcoRI DNA

We used cyclization kinetics experiments and Monte Carlo simulations to determine a structural model for a DNA decamer containing the EcoRI restriction site. Our findings agree well with recent crystal and NMR structures of the EcoRI dodecamer, where an overall bend of seven degrees is distributed symmetrically over the molecule. Monte Carlo simulations indicate that the sequence has a higher flexibility, assumed to be isotropic, compared to that of a "generic" DNA sequence. This model was used as a starting point for the investigation of the effect of cytosine methylation on DNA bending and flexibility. While methylation did not affect bend magnitude or direction, it resulted in a reduction in bending flexibility and under-winding of the methylated nucleotides. We demonstrate that our approach can augment the understanding of DNA structure and dynamics by adding information about the global structure and flexibility of the sequence. We also show that cyclization kinetics can be used to study the properties of modified nucleotides.