Mammalian DNA enriched for replication origins is enriched for snap-back sequences

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA, extruded DNA enriched for replication origins was obtained and denatured. Snap-back DNA, single-stranded DNA with inverted repeats (palindromic sequences), reassociates rapidly into stem-loop structures with zero-order kinetics when conditions are changed from denaturing to renaturing, and can be assayed by chromatography on hydroxyapatite. Origin-enriched nascent DNA strands from mouse, rat and monkey cells growing either synchronously or asynchronously were purified and assayed for the presence of snap-back sequences. The results show that origin-enriched DNA is also enriched for snap-back sequences, implying that some origins for mammalian DNA replication contain or lie near palindromic sequences.     

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.     

Mutations in Pol1 Increase the Mitotic Instability of Tandem Inverted Repeats in Saccharomyces Cerevisiae

Tandem inverted repeats (TIRs or hairpins) of 30 and 80 base-pair unit lengths are unstable mitotically in yeast (Saccharomyces cerevisiae). TIR instability results from deletions that remove part or all of the presumed hairpin structure from the chromosome. At least one deletion endpoint is always at or near the base of the hairpin, and almost all of the repaired junctions occur within short direct sequence repeats of 4 to 9 base pairs. The frequency of this event, which we call ``hairpin excision,' is influenced by chromosomal position, length of the inverted repeats, and the distance separating the repeat units; increasing the distance between the inverted repeats as little as 25 base pairs increases their chromosomal stability. The frequency of excision is not affected by representative rad mutations, but is influenced by mutations in certain genes affecting DNA synthesis. In particular, mutations in POL1/CDC17, the gene that encodes the large subunit of DNA polymerase I, increase the frequency of hairpin deletions significantly, implicating this protein in the normal maintainance of genomic TIRs.     

On the Deletion of Inverted Repeated DNA in Escherichia Coli: Effects of Length, Thermal Stability, and Cruciform Formation in Vivo

We have studied the deletion of inverted repeats cloned into the EcoRI site within the CAT gene of plasmid pBR325. A cloned inverted repeat constitutes a palindrome that includes both EcoRI sites flanking the insert. In addition, the two EcoRI sites represent direct repeats flanking a region of palindromic symmetry. A current model for deletion between direct repeats involves the formation of DNA secondary structure which may stabilize the misalignment between the direct repeats during DNA replication. Our results are consistent with this model. We have analyzed deletion frequencies for several series of inverted repeats, ranging from 42 to 106 bp, that were designed to form cruciforms at low temperatures and at low superhelical densities. We demonstrate that length, thermal stability of base pairing in the hairpin stem, and ease of cruciform formation affect the frequency of deletion. In general, longer palindromes are less stable than shorter ones. The deletion frequency may be dependent on the thermal stability of base pairing involving approximately 16-20 bp from the base of the hairpin stem. The formation of cruciforms in vivo leads to a significant increase in the deletion frequency. A kinetic model is presented to describe the relationship between the physical-chemical properties of DNA structure and the deletion of inverted repeats in living cells.     

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.     

Identification of the replication terminator protein binding sites in the terminus region of the Bacillus subtilis chromosome and stoichiometry of the binding

DNase I footprinting of the interaction between the replication terminator protein (RTP) of Bacillus subtilis and the inverted repeat region (IRR) at the chromosome terminus, to which it binds to block the clockwise replication fork, showed that two major regions of 41 base pairs (bp) were protected from cleavage. These regions corresponded approximately to the imperfect inverted repeats (IRI and IRII) identified previously. Band retardation analyses of the interaction between RTP and portions of the IRR established that each inverted repeat (IRI or IRII) contained two RTP binding sites. By sedimentation equilibrium in the ultracentrifuge, RTP was found to exist as a dimer of 29 kDa at neutral pH and concentrations above 0.2 g/l. Quantitative studies of the RTP-IRR interaction using [3H]RTP and [32P]IRR showed that the fully saturated complex contained eight RTP monomers per IRR. It is concluded that a dimer of RTP binds to each of the four sites in IRR. The apparent dissociation constant for the interaction was estimated (in the presence of 50% glycerol) to be 1.2 x 10(-11) M (dimer of RTP). Glycerol was found to have a marked effect on the affinity of RTP for the IRR and on the relative amounts of the interaction complexes formed; in the absence of glycerol the dissociation constant was approximately 50-fold higher and there was pronounced co-operative binding of RTP dimers to adjacent sites in each inverted repeat. Examination of the DNA sequence in IRI and IRII identified two 8 bp direct repeats in each. The regions protected from DNase I cleavage in each inverted repeat and the protection afforded by a core sequence spanning just one of the 8 bp direct repeats were consistent with each 8 bp repeat representing a recognition sequence for the RTP dimer. A model describing the binding of RTP to the IRR is presented.     

Characteristics of palindromic sequences in DNA of the sea urchin Strongylocentrotus intermedius

Some properties of the palindromic sequences in the sea urchin Strongylocentrotus intermedius nuclear DNA have been studied. It was shown that the amount of "foldback HAP bound DNA" and the S1 nuclease resistant DNA depends on renaturation temperature and Na+ concentration in solution. The authentic fraction of inverted repeats comprises 10-15% of the total DNA. The complexity of the palindromic fraction is approximately 8,2 X 10(7) nucleotide pairs and the average number of inverted repeats approximates 5 X 10(5) per haploid genome. The renaturation kinetics of inverted repeats with excess of total homologous DNA indicates that these sequences are enriched with unique DNA. The possible function of palindromic sequences is discussed.     

Characterization of inverted repeated sequences in wheat nuclear DNA.

The properties of inverted repeated sequences in wheat nuclear DNA have been studied by HAP(1) chromatography, nuclease S1 digestion and electron microscopy. Inverted repeated sequences comprise 1.7% of wheat genome. The HAP studies show that the amount of "foldback HAP bound DNA" depends on DNA length. Inverted repeats appear to be clustered with an average intercluster distance of 25 kb. It is estimated that there are approximately 3 x 10(6) inverted repeats per haploid wheat genome. The sequences around inverted repeats involve all families of repetition frequencies. Inverted repeats are observed as hairpins in electron microscopy. 20% of hairpins are terminated by a single-stranded spacer ranging from 0.3 to 1.5 kb in length. Duplex regions of the inverted repeats range from 0.1 to 0.45 kb with number average values of 0.24 kb and 0.18 kb for unlooped and looped hairpin respectively. Thermal denaturations and nuclease S1 digestions have revealed a length of about 100 bases for duplex regions. The methods used to study inverted repeated sequences are compared and discussed.     

Direct and Inverted Repeats Elicit Genetic Instability by Both Exploiting and Eluding DNA Double-Strand Break Repair Systems in Mycobacteria

Repetitive DNA sequences with the potential to form alternative DNA conformations, such as slipped structures and cruciforms, can induce genetic instability by promoting replication errors and by serving as a substrate for DNA repair proteins, which may lead to DNA double-strand breaks (DSBs). However, the contribution of each of the DSB repair pathways, homologous recombination (HR), non-homologous end-joining (NHEJ) and single-strand annealing (SSA), to this sort of genetic instability is not fully understood. Herein, we assessed the genome-wide distribution of repetitive DNA sequences in the Mycobacterium smegmatis, Mycobacterium tuberculosis and Escherichia coli genomes, and determined the types and frequencies of genetic instability induced by direct and inverted repeats, both in the presence and in the absence of HR, NHEJ, and SSA. All three genomes are strongly enriched in direct repeats and modestly enriched in inverted repeats. When using chromosomally integrated constructs in M. smegmatis, direct repeats induced the perfect deletion of their intervening sequences ∼1,000-fold above background. Absence of HR further enhanced these perfect deletions, whereas absence of NHEJ or SSA had no influence, suggesting compromised replication fidelity. In contrast, inverted repeats induced perfect deletions only in the absence of SSA. Both direct and inverted repeats stimulated excision of the constructs from the attB integration sites independently of HR, NHEJ, or SSA. With episomal constructs, direct and inverted repeats triggered DNA instability by activating nucleolytic activity, and absence of the DSB repair pathways (in the order NHEJ>HR>SSA) exacerbated this instability. Thus, direct and inverted repeats may elicit genetic instability in mycobacteria by 1) directly interfering with replication fidelity, 2) stimulating the three main DSB repair pathways, and 3) enticing L5 site-specific recombination.     

Unstable heterozygosity in a diploid region of herpes simplex virus DNA

We have examined the behavior of a herpes simplex virus strain KOS isolate in which the two inverted repeats flanking the short segment of viral DNA differ in length by approximately 60 base pairs. We find that individual viral DNA molecules exist which contain the two distinguishable repeats, demonstrating that heterology between the repeats is tolerated. However, viruses heterozygous for the two different repeats are unstable, segregating both classes of homozygotes at a high frequency. We propose that this segregation is a consequence of the high-frequency recombination events which also result in genome segment inversion.     

Twelve 43-base-pair repeats map in a cis-acting region essential for partition of plasmid mini-F.

The nucleotide sequence of the DNA region involved in partitioning of plasmid mini-F has been determined. The sequence consists of 12 direct tandemly arranged repeats of 43 base pairs (the two flanking repeats, 43 plus 1 base pairs) with extensive homology to each other. Each repeat contains an additional inverted repeat of 7 base pairs.     

Site-specific deletion at the replication origin of the antibiotic resistance factor R1

Summary The recombinant plasmid pRK101 carrying the complete replication origin of the antibiotic resistance factor R1 suffers frequently a deletion of 218 base pairs, removing parts or all of the origin sequence. This deletion seems to occur always when the Pst-E fragment carrying the replication origin is inserted into the cloning vector pBR322 in an orientation where the direction of R1 replication is the same as that of the vector plasmid and frequently when it is inserted in the opposite direction. DNA sequence analysis around the junction site generated by the deletion in three independently isolated deletion mutants reveals that the deletion occurs at a specific site, namely the end of a 22 bp sequence which is repeated almost identically at the other end of a segment of 197 bp. During the deletion one repeat unit is removed whereas the other is retained. The DNA sequence included by the two repeats contains high symmetric structures, i.e. inverted repeats, direct repeats and palindromes which may represent regulatory sites of the origin.     

Classes of autonomously replicating sequences are found among early-replicating monkey DNA

Thirteen new independent clones of origin-enriched sequences (ors) that are capable of autonomous replication have been identified from a library of 100 ors i clones that had been previously isolated from early replicating monkey (CV-1) DNA. Autonomous replication was assayed by transient episomal replication in transfected HeLa cells; ors-plasmid DNA was isolated at various times after transfection and screened by the DpnI resistance assay and the bromodeoxyuridine (BrdUrd) substitution assay to differentiate between input and newly replicated DNA. Four of the autonomously replicating clones were identified by screening the ors-library with probes of ors 3, 8, 9 and 12, previously shown to be capable of autonomous replication (Frappier and Zannis-Hadjopoulos, Proc. Natl. Acad. Sci. USA (1987) 84, 6668-6672). The other nine functional ors clones were identified among 18 randomly chosen ones, which were similarly screened for autonomous replication. Nucleotide sequence analyses of 11 of the newly identified functional ors plasmids revealed, in most of them, features similar to those present in other viral or eukaryotic replication origins, notably the presence of AT-rich regions and inverted repeats. Pairwise comparisons between the newly identified ors showed no extensive sequence homologies, other than the presence of the alpha-satellite repetitive sequence family in three ors and of the repetitive Alu sequence family in one ors. The results suggest that there exist different classes of mammalian replication origin, highly or moderately repetitive and unique, and that their activation is most probably dependent on the presence of structural determinants rather than on a particular sequence.     

Relation between changes in the palindromic fraction and DNA replication during early stages of sea urchin development

Inverted repeat DNA sequences during embryogenesis were tested by comparing the bulk inverted repeat taken from Strongylocentrotus intermedius sea urchin embryos at different stages of development. This fraction exhibited quantitative and qualitative changes. A reversible quantitative decrease was associated with the 16-cell embryo and blastula stages. Sizing on 1.5% agarose gel indicated that the length of the palindromic sequences at the early blastula stage was predominantly about 200 b. p., and at the pluteus stage 240 b. p. Sensitivity of the palindromic sequences to S1 nuclease digestion at the blastula and gastrula stages was different. It was shown that a specific set of the inverted repeats was included in fragments of DNA--comparising the origin of replication. The results suggest that the change of inverted repeats may be determined by replication processes.     

Structural plasmid instability in Bacillus subtilis: Effect of direct and inverted repeats

Summary Using precise excision as a model system, we have quantified the effect of direct repeats, inverted repeats and the size of the spacer between the repeats in the process of deletion formation in Bacillus subtilis. Both in the presence and absence of inverted repeats, the frequency of precise excision was strongly dependent on the direct repeat length. By increasing the direct repeat length from 9 bp to 18 and 27 bp, the precise excision frequency was raised by 3 and 4 orders of magnitude, respectively. In addition, irrespective of the direct repeat length, the presence of flanking inverted repeats enhanced the excision frequency by 3 orders of magnitude. Varying the inverted repeat length and the spacer size over a wide range did not significantly affect the excision frequencies. These results fit well into a model for deletion formation by slipped mispairing during replication of single-stranded plasmid DNA.     

Nucleotide sequence of the maize transposable element Mul

A cloned DNA fragment from the maize allele Adhl-S3034 contains all of Mul, an insertion element involved in Robertson's Mutator activity. The element is 1367 base pairs (bp) long and is flanked by nine bp direct repeats of insertion site DNA. It has inverted terminal repeats of 215 and 213 bp showing 95% homology. Within the element are two direct repeats of 104 bp showing 96% homology. Four open reading frames (ORFs) were found, two in each DNA strand. Mul can be divided into two halves, each containing one terminal inverted repeat, an internal direct repeat, and two overlapping ORFs. The GC content of each half is high (70%), while that of a central 60 base portion of the element is low (26%). The central region contains the only sequence resembling the TAATA Goldberg and Hogness eukaryotic promoter signal. Multiple copies of DNA sequences related to Mul found in Mutator maize plants are generally similar in organization to the cloned element. A larger version containing a discrete 300 to 400 base pair insertion was found in some Mutator lines.     

Functions of the sequences at the ends of the inverted repeats of pseudorabies virus.

Two mutants were constructed to explore the functions of the sequences at the end of the S terminus of pseudorabies virus (PrV). In mutant vYa, 17 bp from the internal inverted repeat, as well as adjacent sequences from the L component, were deleted. In mutant v135/9, 143 bp from the internal inverted repeat (including sequences with homology to the pac-1 site of herpes simplex virus), as well as adjacent sequences from the L component, were deleted. Our aim in constructing these mutants was to ascertain whether equalization of the terminal regions of the S component would occur, whether genome termini that lack either the terminal 17 or 143 bp would be generated as a result of equalization of the repeats (thereby identifying the terminal nucleotides that may include cleavage signals), and whether inversion of the S component would occur (thereby ascertaining the importance of the deleted sequences in this process). The results obtained show the following (i) The removal of the terminal 17 or 143 bp of the internal S component, including the sequences with homology to the pac-1 site, does not affect the inversion of the Us. (ii) The equalization of both the vYa and the v135/9 inverted repeats occurs at high frequency, the terminal repeats being converted and becoming similar to the mutated internal inverted repeat. (iii) Mutants in which the 17 terminal base pairs (vYa) have been replaced by unrelated sequences are viable. However, the 143 terminal base pairs appear to be essential to virus survival; concatemeric v135/9 DNA with equalized, mutant-type, inverted repeats accumulates, but mature virions with such equalized repeats are not generated at high frequency. Since concatemeric DNA missing the 143 bp at both ends of the S component is not cleaved, the terminal 143 bp that include the sequences with homology to the pac-1 site are necessary for efficient cleavage. (iv) v135/9 intracellular DNA is composed mainly of arrays in which one S component (with two equalized inverted repeats both having the deletion) is bracketed by two L components in opposite orientations and in which two L components are in head-to-head alignment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of nascent monkey DNA synthesized early in the cell cycle.

To study the structure and complexity of animal cell replication origins, we have isolated and cloned nascent DNA from the onset of S phase as follows: African green monkey kidney cells arrested in G1 phase were serum stimulated in the presence of the DNA replication inhibitor aphidicolin. After 18 h, the drug was removed, and DNA synthesis was allowed to proceed in vivo for 1 min. Nuclei were then prepared, and DNA synthesis was briefly continued in the presence of Hg-dCTP. The mercury-labeled nascent DNA was purified in double-stranded form by extrusion (M. Zannis-Hadjopoulos, M. Perisco, and R. G. Martin, Cell 27:155-163, 1981) followed by sulfhydryl-agarose affinity chromatography. Purified nascent DNA (ca. 500 to 2,000 base pairs) was treated with mung bean nuclease to remove single-stranded ends and inserted into the NruI site of plasmid pBR322. The cloned fragments were examined for their time of replication by hybridization to cellular DNA fractions synthesized at various intervals of the S phase. Among five clones examined, four hybridized preferentially with early replicating fractions.