DNA supercoiling enables the type IIS restriction enzyme BspMI to recognise the relative orientation of two DNA sequences

Many proteins can sense the relative orientations of two sequences at distant locations in DNA: some require sites in inverted (head-to-head) orientation, others in repeat (head-to-tail) orientation. Like many restriction enzymes, the BspMI endonuclease binds two copies of its target site before cleaving DNA. Its target is an asymmetric sequence so two sites in repeat orientation differ from sites in inverted orientation. When tested against supercoiled plasmids with two sites 700 bp apart in either repeated or inverted orientations, BspMI had a higher affinity for the plasmid with repeated sites than the plasmid with inverted sites. In contrast, on linear DNA or on supercoiled DNA with sites 1605 bp apart, BspMI interacted equally with repeated or inverted sites. The ability of BspMI to detect the relative orientation of two DNA sequences thus depends on both the topology and the length of the intervening DNA. Supercoiling may restrain the juxtaposition of sites 700 bp apart to a particular alignment across the superhelical axis, but the juxtaposition of sites in linear DNA or far apart in supercoiled DNA may occur without restraint. BspMI can therefore act as a sensor of the conformational dynamics of supercoiled DNA.     

Functional analysis of a centromere from fission yeast: a role for centromere-specific repeated DNA sequences.

A circular minichromosome carrying functional centromere sequences (cen2) from Schizosaccharomyces pombe chromosome II behaves as a stable, independent genetic linkage group in S. pombe. The cen2 region was found to be organized into four large tandemly repeated sequence units which span over 80 kilobase pairs (kb) of untranscribed DNA. Two of these units occurred in a 31-kb inverted repeat that flanked a 7-kb central core of nonhomology. The inverted repeat region had centromere function, but neither the central core alone nor one arm of the inverted repeat was functional. Deletion of a portion of the repeated sequences that flank the central core had no effect on mitotic segregation functions or on meiotic segregation of a minichromosome to two of the four haploid progeny, but drastically impaired centromere-mediated maintenance of sister chromatid attachment in meiosis I. This requirement for centromere-specific repeated sequences could not be satisfied by introduction of random DNA sequences. These observations suggest a function for the heterochromatic repeated DNA sequences found in the centromere regions of higher eucaryotes.     

DNA sequence analysis of a 5.27-kb direct repeat occurring adjacent to the regions of S-episome homology in maize mitochondria.

The DNA sequence of the 5270-bp repeated DNA element from the mitochondrial genome of the fertile cytoplasm of maize has been determined. The repeat is a major site of recombination within the mitochondrial genome and sequences related to the R1(S1) and R2(S2) linear episomes reside immediately adjacent to the repeat. The terminal inverted repeats of the R1 and R2 homologous sequences form one of the two boundaries of the repeat. Frame-shift mutations have introduced 11 translation termination codons into the transcribed S2/R2 URFI gene. The repeated sequence, though recombinantly active, appears to serve no biological function.     

Initiation and termination of DNA transfer during conjugation of IncI1 plasmid R64: roles of two sets of inverted repeat sequences within oriT in termination of R64 transfer

Intercellular transfer of plasmid DNA during bacterial conjugation initiates and terminates at a specific origin of transfer, oriT. We have investigated the oriT structure of conjugative plasmid R64 with regard to the initiation and termination of DNA transfer. Using recombinant plasmids containing two tandemly repeated R64 oriT sequences with or without mutations, the subregions required for initiation and termination were determined by examining conjugation-mediated deletion between the repeated oriTs. The oriT subregion required for initiation was found to be identical to the 44-bp oriT core sequence consisting of two units, the conserved nick region sequence and the 17-bp repeat A sequence, that are recognized by R64 relaxosome proteins NikB and NikA, respectively. In contrast, the nick region sequence and two sets of inverted repeat sequences within the 92-bp minimal oriT sequence were required for efficient termination. Mutant repeat A sequences lacking NikA-binding ability were found to be sufficient for termination, suggesting that the inverted repeat structures are involved in the termination process. A duplication of the DNA segment between the repeated oriTs was also found after mobilization of the plasmid carrying initiation-deficient but termination-proficient oriT and initiation-proficient but termination-deficient oriT, suggesting that the 3' terminus of the transferred strand is elongated by rolling-circle-DNA synthesis.     

S-adenosyl methionine prevents promiscuous DNA cleavage by the EcoP1I type III restriction enzyme

DNA cleavage by the type III restriction endonuclease EcoP1I was analysed on circular and catenane DNA in a variety of buffers with different salts. In the presence of the cofactor S-adenosyl methionine (AdoMet), and irrespective of buffer, only substrates with two EcoP1I sites in inverted repeat were susceptible to cleavage. Maximal activity was achieved at a Res2Mod2 to site ratio of approximately 1:1 yet resulted in cleavage at only one of the two sites. In contrast, the outcome of reactions in the absence of AdoMet was dependent upon the identity of the monovalent buffer components, in particular the identity of the cation. With Na+, cleavage was observed only on substrates with two sites in inverted repeat at elevated enzyme to site ratios (>15:1). However, with K+ every substrate tested was susceptible to cleavage above an enzyme to site ratio of approximately 3:1, including a DNA molecule with two directly repeated sites and even a DNA molecule with a single site. Above an enzyme to site ratio of 2:1, substrates with two sites in inverted repeat were cleaved at both cognate sites. The rates of cleavage suggested two separate events: a fast primary reaction for the first cleavage of a pair of inverted sites; and an order-of-magnitude slower secondary reaction for the second cleavage of the pair or for the first cleavage of all other site combinations. EcoP1I enzymes mutated in either the ATPase or nuclease motifs did not produce the secondary cleavage reactions. Thus, AdoMet appears to play a dual role in type III endonuclease reactions: Firstly, as an allosteric activator, promoting DNA association; and secondly, as a "specificity factor", ensuring that cleavage occurs only when two endonucleases bind two recognition sites in a designated orientation. However, given the right conditions, AdoMet is not strictly required for DNA cleavage by a type III enzyme.     

A map of the restriction targets in yeast 2 micron plasmid DNA cloned on bacteriophage lambda

Summary The 2 micron circular DNA from S. cerevisiae has been cloned on bacteriophage . The two forms of circular DNA which exist in equilibrium due to recombination between inverted repeat sequences were separated as stable clones, and a map of the targets for restriction endonucleases EcoRI, HindIII and HpaI was constructed. The circular DNAs isolated from a particular oligomycin resistant strain and its parent oligomycin sensitive strain were compared by restriction endonuclease analysis, and no difference was detected. The potential uses of cloned 2 micron DNA in determining the possible biological role of these plasmids are considered.     

Inverted repeated sequences in yeast nuclear DNA.

The inverted repeated sequences (foldback DNA) of yeast nuclear DNA have been examined by electron microscopy and hydroxyapatite chromatography. Of the inverted repeat structures seen in the electron microscope, 34% were hairpins and 66% had a single stranded loop at the end of a duplex stem. The number average length of the repeat was 0.3 kb and the single stranded loop was 1.6 kb. It is estimated that there are approximately 250 inverted repeats per haploid genome. A statistical analysis of the frequency of molecules containing multiple inverted repeats showed that these sequences are non-randomly distributed. The distribution of inverted repeats was also examined by measuring the fraction of total DNA in the foldback fraction that bound to hydroxyapatite as a function of single strand fragment size. This analysis also indicated that the inverted repeats are clustered. Renaturation kinetic analysis of isolated foldback and inverted repeat stem sequence DNA showed that these sequences are enriched for repetitive DNA.     

Physical maps for Herpes simplex virus type 1 DNA for restriction endonucleases Hind III, Hpa-1, and X. bad.

It has been proposed that the genome of herpes simplex virus type 1 (HSV-1) consists of two internal unique sequences, S and L, bounded by two sets of redundant sequences (P. Sheldrick and N. Berthelot, 1974). In this arrangement, terminal sequences (TRs and TRl) are repeated in an internal inverted form (IRs and IRl) and delimit S and L. Furthermore, a body of evidence has accumulated that suggests that S and L themselves are inverted, giving rise to four related forms of the HSV genome. In this study the ordering of restruction endonuclease fragments of HSV-1 DNA for physical maps has been studied using molecular hybridization techniques and the cleavage of isolated restriction endonuclease fragments with further restriction endonucleases. Physical maps for the fragments produced by Hind III, Hpa-1, and X. bad have been constructed for the four related forms of the HSV-1 genome. TRs and IRs were found to be between 3.5 x 10(6) and 4.5 x 10(6) daltons, TRl and IRl about 6 x 10(6) daltons, S about 8 x 10(6) to 9 x 10(6) daltons, and L about 6.8 x 10(6) daltons.     

Structure of the joint region and the termini of the DNA of herpes simplex virus type 1.

Analysis of restriction endonuclease cleavage sites within the inverted, repeated sequences in the joint region of the DNA of herpes simplex virus type 1 strain KOS revealed the presence of two types of sequence heterogeneity. The first was an insertion of 280 base pairs or multiples of 280 base pairs which was found in approximately half of all DNA molecules from every plaque-purified stock of virus. These insertions seemed to be tandem duplications of sequences which were present at the joint and correspond closely to the inverted terminal redunancy. The second type of heterogeneity was due to variable insertions and deletions which were present in some, but not all, plaque-purified virus stocks. Comparison of restriction fragments from the joint region with fragments from the termini indicated that in the simplest observed molecules of herpes simplex virus type 1 DNA, only one copy of the inverted terminal redundancy was present at the joint. A map of restriction endonuclease cleavage sites in the joint region is presented.     

The nuclease domain of adeno-associated virus rep coordinates replication initiation using two distinct DNA recognition interfaces

Integration into a particular location in human chromosomes is a unique property of the adeno-associated virus (AAV). This reaction requires the viral Rep protein and AAV origin sequences. To understand how Rep recognizes DNA, we have determined the structures of the Rep endonuclease domain separately complexed with two DNA substrates: the Rep binding site within the viral inverted terminal repeat and one of the terminal hairpin arms. At the Rep binding site, five Rep monomers bind five tetranucleotide direct repeats; each repeat is recognized by two Rep monomers from opposing faces of the DNA. Stem-loop binding involves a protein interface on the opposite side of the molecule from the active site where ssDNA is cleaved. Rep therefore has three distinct binding sites within its endonuclease domain for its different DNA substrates. Use of these different interfaces generates the structural asymmetry necessary to regulate later events in viral replication and integration.     

The organization of repetitive sequences in a cluster of rabbit β-like globin genes

Several complementary procedures were used to identify and characterize DNA sequences which are repeated within a 44 kilobase (kb) segment of rabbit chromosomal DNA containing four different rabbit β-like globin genes (β1–β4). Cross-hybridization between cloned DNAs from different regions of the gene cluster indicates the presence of a complex array of repeat sequences interspersed with the globin genes. We classified 20 different repeat sequences into five families whose members cross-hybridize. Electron microscopy was used to determine the location, size and relative orientations of many of the repeat sequences. Both direct and inverted repeats were identified, with sizes ranging from 140 to 1400 base pairs (bp). Each of the four closely linked globin genes is flanked by at least one pair of inverted repeats of 140–400 bp, and the entire set of four genes is flanked by an inverted repeat of 1400 bp. Two of the five repeat families contain repeat sequences of different sizes. We found that the smaller sequence elements can occur individually or in association with the larger repeat sequences, suggesting that the larger repeats may be composed of more than one smaller repeat sequence. The restriction fragments containing the intracluster repeats also contain sequences which are repeated many times in total rabbit genomic DNA, but it is not known whether the genomic and intracluster repeats are the same sequences. The results provide the first demonstration of the relationship between single-copy and repetitive DNA sequences in a large segment of chromosomal DNA containing a well characterized set of developmentally regulated genes.     

Formation of a cruciform structure at the simian virus 40 replication origin abolishes T-antigen binding to the origin in vitro.

Heteroduplex DNA molecules were formed by annealing an intact simian virus replication origin-containing fragment to a mutant derivative lacking the indigenous wild-type 27-base-pair (bp) inverted repeat within this structure and containing a nonhomologous 26-bp inverted repeat sequence in its place. Results of restriction enzyme and S1 endonuclease cleavage analyses strongly suggested that a 13-bp stem-loop structure formed at the site of nonhomology between these two DNAs. This structure lies within the boundary of simian virus 40 T-antigen-binding site 2, and its presence inhibited T-antigen binding to that sequence but not to an adjacent higher-affinity binding site (site 1). Therefore, the conformation of sequences within an otherwise intact T-antigen-binding site can have major effects upon T-antigen binding there.     

A small family of elements with long inverted repeats is located near sites of developmentally regulated DNA rearrangement in Tetrahymena thermophila.

Extensive DNA rearrangement occurs during the development of the somatic macronucleus from the germ line micronucleus in ciliated protozoans. The micronuclear junctions and the macronuclear product of a developmentally regulated DNA rearrangement in Tetrahymena thermophila, Tlr1, have been cloned. The intrachromosomal rearrangement joins sequences that are separated by more than 13 kb in the micronucleus with the elimination of moderately repeated micronucleus-specific DNA sequences. There is a long, 825-bp, inverted repeat near the micronuclear junctions. The inverted repeat contains two different 19-bp tandem repeats. The 19-bp repeats are associated with each other and with DNA rearrangements at seven locations in the micronuclear genome. Southern blot analysis is consistent with the occurrence of the 19-bp repeats within pairs of larger repeated sequences. Another family member was isolated. The 19-mers in that clone are also in close proximity to a rearrangement junction. We propose that the 19-mers define a small family of developmentally regulated DNA rearrangements having elements with long inverted repeats near the junction sites. We discuss the possibility that transposable elements evolve by capture of molecular machinery required for essential cellular functions.     

Sequence arrangement in herpes simplex virus type 1 DNA: identification of terminal fragments in restriction endonuclease digests and evidence for inversions in redundant and unique sequences.

It has been proposed by Sheldrick and Berthelot (1974) that the terminal sequences of herpes simplex virus type 1 (HSV-1) DNA are repeated in an internal inverted form and that the inverted redundant sequences delimit and separate two unique sequences, S and L. In this study the sequence arrangement in HSV-1 DNA has been investigated with restriction endonuclease cleavage, end-labeling studies, and molecular hybridization experiments. The terminal fragments in digests with restriction endonucleases Hind III, Hpa-1, EcoRI and Bum were identified and shown to be consistent with the Sheldrick and Berthelot model. Inverted fragments which contain unique sequences as well as redundant sequences, and which the model predicts, were identified by DNA-DNA hybridization studies. Further cleavage of Bum fragments with Hpa-1 also revealed inversions of the terminal sequences that contained unique sequences. The results obtained showed that the unique sequences S and L are relatively inverted in different DNA molecules in the population, resulting in the presence of four related genomes with rearranged sequences in apparently equal amounts. The redundant sequences bounding S do not share complete sequence homology with those bounding L, but hybridization studies are presented which show that the terminal 0.3% of the genome is repeated in every redundant sequence.     

Organisation of inverted repeat sequences in hamster cell nuclear DNA.

Hamster cell nuclear DNA is shown to contain inverted repeat (foldback) sequences, in some respects similar to the foldback fraction in DNA from other animal cell types. Using electron microscopy the majority of foldback duplexes are shown to be located in simple hairpin-like DNA structures, formed from individual pairs of complementary inverted repeated sequences 50--1000 nucleotides in length, in some cases arranged in tandem, and in other cases separated by intervening sequences, up to 16000 nucleotide residues long. In addition, a novel class of foldback structure, referred to as 'bubbled hairpins' is reported, which appear to be formed from clusters of inverted repeat sequences that are separated from adjacent clusters of complementary inverted repeats by large intervening sequences which vary in length from 5000 to over 20000 nucleotide residues. Due to the special pattern of distribution of these latter inverted repeat sequences, 'bubbled hairpins' are observed only in long foldback DNA. Evidence is presented that the distribution of foldback sequences in hamster cell DNA is highly ordered. The lengths of the intervening single chains in foldback structures appear to vary non-randomly. This gives rise to a localised periodic pattern of organisation that is believed to be a consequence of regular alternating arrangements of foldback and non-foldback sequences in the segments of DNA from which foldback structures are derived.     

Colobus type C virus: molecular cloning of unintegrated viral DNA and characterization of the endogenous viral genomes of Colobus.

The unintegrated viral DNA intermediates of colobus type C virus (CPC-1) were isolated from infected human cells that were permissive for viral growth. There were two major species of DNA, linear molecules with two copies of the long terminal repeat and relaxed circles containing only a single long terminal repeat. In addition, there was a minor species (approximately 10%) composed of relaxed circles with two copies of the long terminal repeat. A restriction endonuclease map of the unintegrated DNA was constructed. The three EcoRI fragments of circular CPC-1 DNA were cloned in the EcoRI site of lambda gtWES . lambda B and then subcloned in the EcoRI site of pBR322. Using these subgenomic fragments as probes, we have characterized the endogenous viral sequences found in colobus cellular DNA. They are not organized in tandem arrays, as is the case in some other gene families. The majority of sequences detected in cellular DNA have the same map as the CPC-1 unintegrated DNA at 17 of 18 restriction endonuclease sites. There are, however, other sequences that are present in multiple copies and do not correspond to the CPC-1 map. They do not contain CPC-1 sequences either in an altered form or fused to common nonviral sequences. Instead, they appear to be derived from a distinct family of sequences that is substantially diverged from the CPC-1 family. This second family of sequences, CPC-2, is also different from the sequences related to baboon endogenous type C virus that forms a third family of virus-related sequences in the colobus genome.     

Large inverted duplications are associated with gene amplification

Amplified DNA can be found in arrays of large repeated units, with each repeat unit containing a marker gene and surrounding DNA sequences. Amplified DNA sequences from established cell lines were assessed for the presence of repeat units in the form of inverted duplications. Inverted duplicated DNA was detected by virtue of its concentration-independent resistance to S1 hydrolysis after denaturation and rapid renaturation. Using this assay, inverted duplications were detected in amplified DNA (both DM and HSR configurations) containing the myc gene (16-50 copies/cell) in four human tumor cell lines and in amplified DNA containing the CAD gene (30-200 copies/cell) in three PALA-resistant BHK cell lines. The widespread association of inverted duplications with amplified DNA must bear on the amplification mechanism.     

Anatomy of herpes simplex virus DNA. II. Size, composition, and arrangement of inverted terminal repetitions.

Electron microscope studies on self-annealed intact single strands and on partially denatured molecules show that herpes simplex virus 1 DNA consists of two unequal regions, each bounded by inverted redundant sequences. Thus the region L (70 percent of the contour length of the DNA) separates the left terminal region a1b from its inverted repeat b'a'1, each of which comprises 6 percent of the DNA. The region S (9.4 percent of DNA) separates the right terminal region cas (4.3 percent of the DNA) from its inverted repeat a'sc'. The regions of the two termini which are inverted and repeated itnernally differ in topology. Thus, cas is guanine plus cytosine rich, whereas only the terminal 1 percent of the a1b region, designated as subregion a1, is guanine plus cytosine rich.     

Effect of base composition at the center of inverted repeated DNA sequences on cruciform transitions in DNA

We have analyzed the effect of base composition at the center of symmetry of inverted repeated DNA sequences on cruciform transitions in supercoiled DNA. For this we have constructed two series of palindromic DNA sequences: one set with differing center and one set with differing center and arm sequences. The F series consists of two 96-base pair perfect inverted repeats which are identical except for the central 10 base pairs which consist of pure AT or GC base pairs. The S series was constructed such that the overall base composition of the inverted repeats was identical but in which the positioning of blocks of AT- and GC-rich sequences varied. The rate of cruciform formation for the inverted repeats in plasmid pUC8 was dramatically influenced by the 8-10 base pairs at the center of the inverted repeat. Inverted repeats with 8-10 AT base pairs in the center were kinetically much more active in cruciform formation than inverted repeats with 8-10 GC base pairs in the center. These experiments show a dominant influence of the center sequences of inverted repeats on the rate of cruciform formation.     

Physical map of infectious baboon type C viral DNA and sites of integration in infected cells.

Three species of unintegrated viral DNAs were found in permissive cells infected with baboon type C virus. The major species was a 9.0-kilobase (kb) linear DNA that was infectious. A restriction endonuclease map of this DNA was constructed and oriented with respect to the viral RNA. The linear DNA had a 0.6-kb sequence repeated at each terminus. These terminal repeat sequences were required for infectivity of the viral DNA. The minor species of the unintegrated viral DNAs were covalently closed circles of 9.0 and 8.4 kb. The smaller circle was in two- to threefold excess over the larger circle. The difference appeared to be that the smaller circle lacked one of the two 0.6-kb repeat sequences found in the larger circle. Restriction endonuclease maps of the integrated viral DNAs were constructed, and the sequences on both viral DNA and cellular DNA that are involved in integration were determined. The integrated viral DNA map was identical to that of the unintegrated infectious 9.0-kb linear DNA. Therefore, a specific site in the terminal repeat sequence of the viral DNA was used to integrate with the host cell DNA. The sizes of the cellular DNA fragments were different from clone to clone but stable with cell passage. Therefore, many sites in the cell DNA can recombine with the viral DNA.