Repair by recombination of DNA containing a palindromic sequence

We report here that homologous recombination functions are required for the viability of Escherichia coli cells maintaining a 240 bp chromosomal inverted repeat (palindromic) sequence. Wild-type cells can successfully replicate this palindrome but recA , recB or recC mutants carrying the palindrome are unviable. The dependence on homologous recombination for cell viability is overcome in sbcC mutants. Directly repeated copies of the DNA containing the palindrome are rapidly resolved to single copies in wild-type cells but not in sbcC mutants. Our results suggest that double-strand breaks introduced at the palindromic DNA sequence by the SbcCD nuclease are repaired by homologous recombination. The repair is conservative and the palindrome is retained in the repaired chromosome. We conclude that SbcCD can attack secondary structures but that repair conserves the DNA sequence with the potential to fold.     

Nucleosome phasing on a DNA fragment from the replication origin of simian virus 40 and rephasing upon cruciform formation of the DNA.

Nucleosomes were reconstituted in vitro from a fragment of DNA spanning the simian virus 40 minimal replication origin. The fragment contains a 27-base-pair palindrome (perfect inverted repeat). DNA molecules with stable cruciform structures were generated by heteroduplexing this DNA fragment with mutants altered within the palindromic sequence (C. Nobile and R. G. Martin, Int. Virol., in press). Analyses of the structural features of the reconstituted nucleosomes by the DNase I footprint technique revealed two alternative DNA-histone arrangements, each one accurately phased with respect to the uniquely labeled DNA ends. As linear double-stranded DNA, a unique core particle was formed in which the histones strongly protected the regions to both sides of the palindrome. The cruciform structure seemed to be unable to associate with core histones and, therefore, an alternative phasing of the histone octamer along the DNA resulted. Thus, nucleosome positioning along a specific DNA sequence appears to be influenced in vitro by the secondary structure (linear or cruciform) of the 27-base-pair palindrome. The formation of cruciform structures in vivo, if they occur, might therefore represent a molecular mechanism by which nucleosomes are phased.     

In vivo loss of supercoiled DNA carrying a palindromic sequence

Summary Interest in the fate of long palindromic DNA sequences in E. coli has been kindled by the observation that their inviability is overcome in recBC sbcB strains and that these hosts permit the construction of DNA libraries containing long palindromic sequences present in the human genome. In this paper we show that a reduction in the level of intracellular supercoiled DNA occurs as the result of the presence of a 530 bp palindrome in bacteriophage lambda. This reduction occurs in Rec⁺ and recA strains but not in strains lacking exonucleases V and I (recBC sbcB). However, the DNA must be active (not repressed) for this reduction to be observed, since it is not seen in a Rec⁺ host lysogenic for phage lambda. These results argue against two hypotheses: firstly, that the palindrome causes inviability solely by interfering with packaging, and secondly, that it dose so solely by interfering with recombination. Conversely, these results suggest that a feature of active monomeric DNA (probably its replication) is involved in inviability.     

Symmetry elements in DNA structure important for recognition/methylation by DNA [amino]-methyltransferases

The phage T4Dam and EcoDam DNA-[adenine-N6] methyltransferases (MTases) methylate GATC palindromic sequences, while the BamHI DNA-[cytosine-N4] MTase methylates the GGATCC palindrome (which contains GATC) at the internal cytosine residue. We compared the ability of these enzymes to interact productively with defective duplexes in which individual elements were deleted on one chain. A sharp decrease in k_cat was observed for all three enzymes if a particular element of structural symmetry was disrupted. For the BamHI MTase, integrity of the ATCC was critical, while an intact GAT sequence was necessary for the activity of T4Dam, and an intact GA was necessary for EcoDam. Theoretical alignment of the region of best contacts between the protein and DNA showed that in the case of a palindromic interaction site, a zone covering the 5′-symmetric residues is located in the major groove versus a zone of contact covering the 3′-symmetric residues in the minor groove. Our data fit a simple rule of thumb that the most important contacts are aligned around the methylation target base: if the target base is in the 5′ half of the palindrome, the interaction between the enzyme and the DNA occurs mainly in the major groove; if it is in the 3′ half, the interaction occurs mainly in the minor groove.     

Palindrome resolution and recombination in the mammalian germ line.

Genetic instability is promoted by unusual sequence arrangements and DNA structures. Hairpin DNA structures can form from palindromes and from triplet repeats, and they are also intermediates in V(D)J recombination. We have measured the genetic stability of a large palindrome which has the potential to form a one-stranded hairpin or a two-stranded cruciform structure and have analyzed recombinants at the molecular level. A palindrome of 15.3 kb introduced as a transgene was found to be transmitted at a normal Mendelian ratio in mice, in striking contrast to the profound instability of large palindromes in prokaryotic systems. In a significant number of progeny mice, however, the palindromic transgene is rearranged; between 15 and 56% of progeny contain rearrangements. Rearrangements within the palindromic repeat occur both by illegitimate and homologous, reciprocal recombination. Gene conversion within the transgene locus, as quantitated by a novel sperm fluorescence assay, is also elevated. Illegitimate events often take the form of an asymmetric deletion that eliminates the central symmetry of the palindrome. Such asymmetric transgene deletions, including those that maintain one complete half of the palindromic repeat, are stabilized so that they cannot undergo further illegitimate rearrangements, and they also exhibit reduced levels of gene conversion. By contrast, transgene rearrangements that maintain the central symmetry continue to be unstable. Based on the observed events, we propose that one mechanism promoting the instability of the palindrome may involve breaks generated at the hairpin structure by a hairpin-nicking activity, as previously detected in somatic cells. Because mammalian cells are capable of efficiently repairing chromosome breaks through nonhomologous processes, the resealing of such breaks introduces a stabilizing asymmetry at the center of the palindrome. We propose that the ability of mammalian cells to eliminate the perfect symmetry in a palindromic sequence may be an important DNA repair pathway, with implications regarding the metabolism of palindromic repeats, the mutability of quasipalindromic triplet repeats, and the early steps in gene amplification events.     

Local DNA Sequence Control of Deletion Formation in Escherichia coli Plasmid Pbr322

The specificity of deletion formation was studied using tests involving reversion of palindromic insertion mutations. Insertions of a Tn5-related transposon at 13 sites in the ampicillin-resistance (amp) gene of plasmid pBR322 were shortened to a nested set of perfect palindromes, 22, 32 and 90 bp long. We monitored frequencies of reversion to Ampr, which is the result of deletion of the palindrome plus one copy of the flanking 9 bp direct repeats (which had been formed by transposition). Revertant frequencies were found to depend on the location and the sequence of the palindromic insert. Changing a 45-kb interrupted palindrome to a 22-bp perfect palindrome stimulated deletion formation by factors of from fourfold to 545-fold among the 13 sites, while elongation of the perfect palindrome from 22 to 90 bp stimulated deletion formation by factors of from eight- to 18,000-fold. We conclude that deletion formation is strongly affected by subtle features of DNA sequence or conformation, both inside and outside the deleted segment, and that these effects may reflect specific interactions of DNA processing proteins with template DNAs.     

Palindrome regeneration by template strand-switching mechanism at the origin of DNA replication of porcine circovirus via the rolling-circle melting-pot replication model

Palindromic sequences (inverted repeats) flanking the origin of DNA replication with the potential of forming single-stranded stem-loop cruciform structures have been reported to be essential for replication of the circular genomes of many prokaryotic and eukaryotic systems. In this study, mutant genomes of porcine circovirus with deletions in the origin-flanking palindrome and incapable of forming any cruciform structures invariably yielded progeny viruses containing longer and more stable palindromes. These results suggest that origin-flanking palindromes are essential for termination but not for initiation of DNA replication. Detection of template strand switching in the middle of an inverted repeat strand among the progeny viruses demonstrated that both the minus genome and a corresponding palindromic strand served as templates simultaneously during DNA biosynthesis and supports the recently proposed rolling-circle "melting-pot" replication model. The genome configuration presented by this model, a four-stranded tertiary structure, provides insights into the mechanisms of DNA replication, inverted repeat correction (or conversion), and illegitimate recombination of any circular DNA molecule with an origin-flanking palindrome.     

Statistical significance of symmetrical and repetitive segments in DNA.

Methods of computer analysis for the recurrence of symmetrical and repetitive elements in large numbers of DNA sequences are described, together with derivations of appropriate quantitative criteria for the evaluation of the statistical significance of these elements in DNAs of different base composition. Examples of some extraordinary variations in the occurrence of symmetrical and repetitive elements are provided, many of which are new. Special consideration is devoted to a determination of the statistical significance of a two-fold palindrome at the origin of replication. A computer search of 14 independently determined DNA sequences containing an origin of replication locus indicates each contains a large two-fold palindrome. The average length of this palindrome is 28 +/- 6 base pairs, of which 22 contribute to the palindromic symmetry. The probability of occurrence of such a palindrome is only 1/26000, while the probability of occurrence in all 14 different species is (1/26000).     

Detection of template strand switching during initiation and termination of DNA replication of porcine circovirus

Nucleotide substitution mutagenesis was conducted to investigate the importance of the inverted repeats (palindrome) at the origin of DNA replication (Ori) of porcine circovirus type 1 (PCV1). Viral genomes with engineered mutations on either arm or both arms of the palindrome were not impaired in protein synthesis and yielded infectious progeny viruses with restored or new palindromes. Thus, a flanking palindrome at the Ori was not essential for initiation of DNA replication, but one was generated inevitably at termination. Among the 26 viruses recovered, 16 showed evidence of template strand switching, from minus-strand genome DNA to palindromic strand DNA, during biosynthesis of the Ori. Here I propose a novel rolling-circle "melting-pot" model for PCV1 DNA replication. In this model, the replicator Rep protein complex binds, destabilizes, and nicks the Ori sequence to initiate leading-strand DNA synthesis. All four strands of the destabilized inverted repeats exist in a "melted" configuration, and the minus-strand viral genome and a palindromic strand are available as templates, simultaneously, during initiation or termination of DNA replication. Inherent in this model is a "gene correction" or "terminal repeat correction" mechanism that can restore mutilated inverted-repeat sequences to a palindrome at the Ori of circular DNAs or at the termini of circularized linear DNAs. Potentially, the melted state of the inverted repeats increases the rate of noncomplementary or illegitimate nucleotide incorporation into the palindrome. Thus, this melting-pot model provides insight into the mechanisms of DNA replication, gene correction, and illegitimate recombination at the Ori of PCV1, and it may be applicable to the replication of other circular DNA molecules.     

A native cruciform DNA structure probed in bacteria by recombinant T7 endonuclease

T7 endonuclease preferentially cleaves purified supercoiled pBR322 and colE1 plasmids at the single-stranded regions exposed when palindromic sequences assume cruciform structures (Panayotatos, N., and Wells, R.D. (1981) Nature 289, 466-470). In vivo, however, induction of nuclease synthesis off a cloned gene caused complete degradation of the bacterial DNA but not of the plasmid vector; presumably, single-stranded regions (cruciforms?) on the genome effectively complete for the nuclease with similar sites on the plasmid (Panayotatos, N., and Fontaine, A. (1985) J. Biol. Chem. 260, 3173-3177). To overcome this competition, we introduced on the plasmid the naturally occurring colE1 palindrome which forms a more stable cruciform in vitro. In addition, we increased the target size (and the T7 endonuclease gene dosage) by raising the copy number of the plasmid 5-fold. Induction of the endonuclease encoded by this new plasmid (pLAT75) resulted not only in degradation of genomic DNA but also in intracellular nicking and linearization of the plasmid. The cleavage site in vivo was mapped at the colE1 palindrome and coincided with the site cleaved specifically in vitro by either T7 or S1 endonuclease only when this palindrome assumes the cruciform structure. These results indicate that cruciform structures exist intracellularly and demonstrate the usefulness of endonucleases as probes of DNA topology in vivo.     

Mutations predetermined by the primary structure of DNA

This study is concerned with an experimental verification of hypotheses postulating the involvement of self-complementary nucleotide sequences in the formation of deletions and insertions. It was suggested that deletions can arise in the regions of self-complementary nucleotide sequences, which allows the formation of the hairpin structures in a single-stranded DNA, arising during excision repair. These hairpin structures can be eliminated by nucleases or during DNA replication. Insertions can arise as a result of homologous recombination, when a migrating DNA strand contains a self-complementary sequence which forms hairpin structure. Model experiments were carried out with the pBR322 plasmid. A plasmid DNA with premutational damage in the palindrome-containing region was constructed by in vitro dimethylsulfate modification of one strand of EcoRI-BamHI restriction fragment. The plasmid was used for transformation of Escherichia coli. Restriction mapping and nucleotide analysis of the mutant DNAs demonstrated that they all contained deletions. The end points of the deletions coincide with the palindrome. To model homologous recombination, a plasmid with D-loop was constructed. A single-stranded DNA fragment containing palindrome forming a hairpin structure was introduced into the plasmid DNA and covalently fixed in the complex. When E. coli cells were transfected with this DNA, plasmid mutants containing insertions predetermined by palindromic structure arose. The evolutionary role of mutations predetermined by primary DNA structure is discussed.     

Modes of DNA replication primed with the oligomer of palindrome

What is the precise molecular mechanism of semi-conservative DNA replication? After the great efforts of the past 20 years, molecular biology has now established the discontinuous syntheses of daughter DNA on both of the parental strands. In order to explain this type of discontinuous replication, we introduce the concept of a palindromic primer.First we focus our attention on various oligomers (RNA or DNA) which appear usually or occasionally in the process of replication. Then we propose the palindromic nature of these oligomers so as to serve as the primer of DNA synthesis. This postulation gives a theoretical reasoning for the discontinuities of both new strands in the fork region of replication.Subsequently we consider Watson's concatemeric intermediate theory, proposed for the explanation of replicative synthesis of phage T7 DNA. By considering the contribution of some sequence-specific endonuclease(s), we suggest the existence of partial palindromic sequences of bases at the connecting region(s) in which the redundant ends of the respective phage DNA molecules are overlapping. Another theory on the replication of linear chromosomal DNA including the concept of the terminal palindromic sequence of bases is also analyzed from the viewpoint of palindromic primer. Further, some recent experimental approaches, especially on the origin(s) of DNA replication, are shown to favour the concept of a palindromic primer.     

A perfect palindrome in the Escherichia coli chromosome forms DNA hairpins on both leading- and lagging-strands

DNA palindromes are hotspots for DNA double strand breaks, inverted duplications and intra-chromosomal translocations in a wide spectrum of organisms from bacteria to humans. These reactions are mediated by DNA secondary structures such as hairpins and cruciforms. In order to further investigate the pathways of formation and cleavage of these structures, we have compared the processing of a 460 base pair (bp) perfect palindrome in the Escherichia coli chromosome with the same construct interrupted by a 20 bp spacer to form a 480 bp interrupted palindrome. We show here that the perfect palindrome can form hairpin DNA structures on the templates of the leading- and lagging-strands in a replication-dependent reaction. In the presence of the hairpin endonuclease SbcCD, both copies of the replicated chromosome containing the perfect palindrome are cleaved, resulting in the formation of an unrepairable DNA double-strand break and cell death. This contrasts with the interrupted palindrome, which forms a hairpin on the lagging-strand template that is processed to form breaks, which can be repaired by homologous recombination.     

Persistence of DNA synthesis arrest sites in the presence of T4 DNA polymerase and T4 gene 32, 44, 45 and 62 DNA polymerase accessory proteins.

DNA synthesis by phage T4 DNA polymerase is arrested at specific sequences in single-stranded DNA templates. To determine whether or not T4 DNA polymerase accessory proteins 32, 44, 45 and 62 eliminated recognition of these arrest sites, unique primer-templates were constructed in which DNA synthesis began at a DNA primer located at different distances from palindromic and nonpalindromic arrest sites. Nucleotide positions that caused polymerase to pause or leave the template were identified by sequence analysis of 5'-end labeled nascent DNA chains. Stable hairpin structures at palindromic sequences were confirmed by acetylation of single-stranded sequences with bromoacetaldehyde. Our results confirmed that these T4 DNA polymerase accessory proteins stimulated T4 DNA polymerase activity and processivity on natural as well as homopolymer primer-templates. However, they did not alter recognition of DNA synthesis arrest sites by T4 DNA polymerase. Extensive DNA synthesis resulted from an increased rate of translocation and/or processivity to the same extent over all DNA sequences.     

Sequences of the Epstein-Barr Virus (EBV) large internal repeat form the center of a 16-kilobase-pair palindrome of EBV (P3HR-1) heterogeneous DNA.

We have previously characterized several genomic rearrangements of Epstein-Barr virus (EBV) DNA contained in one of the defective EBV genomes harbored by the P3HR-1 (HR-1) line (H. B. Jenson, M. S. Rabson, and G. Miller, J. Virol. 58:475-486, 1986). One recombinant clone of heterogeneous DNA (het DNA) from this defective genome is an EcoRI fragment of 16 kilobase pairs (kbp) which is a palindrome. DNA digestion fragments specific for the center of this palindrome were present in cells which contained het DNA but not in cells which lacked het DNA. Thus, the palindrome was not an artifact of DNA cloning. The organization of the center of this palindrome was studied by DNA sequencing. The comparable region of the parental HR-1 genome was also studied by DNA sequencing. The central 3,495 base pairs (bp) of the palindrome were composed of sequences derived exclusively from internal repeat 1 of EBV, represented by BamHI W fragment. At each end of the central 3,495 hp was a symmetrical recombination with sequences of BamHI-Z, located more than 50 kbp away on the standard EBV genome. The central 3,495 bp were composed of an unduplicated 341 bp flanked by two perfect palindromic repeats of 1,577 bp. The 341-bp unique region was a portion of a 387-bp region of standard HR-1 BamHI-W which was identical to the central 387 bp of the palindrome. This central 387-bp region contained numerous stretches of dyad symmetry capable of forming a large stem-and-loop structure. The palindromic rearrangement had created two novel open reading frames in het DNA derived from standard HR-1 BamHI-W sequences. These two het DNA open reading frames had different amino termini but identical carboxy termini derived from the large open reading frame in standard HR-1 BamHI-W (HR-1 BWRF1). The BamHI-W sequences found in het DNA did not include either the TATA box of standard HR-1 BamHI-W or the exons which are present in the potentially polycistronic latent mRNAs encoding EBV nuclear antigens. These marked alterations in genomic structure may relate to the unique biologic properties of virus stocks containing het DNA by creation of new polypeptides or by formation or deletion of regulatory or functional signals.     

Shope fibroma virus DNA topoisomerase catalyses holliday junction resolution and hairpin formation in vitro

The telomeres of poxviral chromosomes comprise covalently closed hairpin structures bearing mismatched bases. These hairpins are formed as concatemeric replication intermediates and are processed into mature, unit-length genomes. The structural transitions and enzymes involved in telomere resolution are poorly understood. Here we show that the type I topoisomerase of Shope fibroma virus (SFV) can promote a recombination reaction which converts cloned SFV replication intermediates into hairpin-ended molecules resembling mature poxviral telomeres. Recombinant SFV topoisomerase linearised a palindromic plasmid bearing 1.5 kb of DNA encoding the SFV concatemer junction, at a site near the centre of inverted-repeat symmetry. Most of these linear reaction products bore hairpin tips as judged by denaturing gel electrophoresis. The resolution reaction required palindromic SFV DNA sequences and was inhibited by compounds which block branch migration (MgCl2) or poxviral topoisomerases. The resolution reaction was also slow, needed substantial quantities of topoisomerase, and required that the palindrome be extruded in a cruciform configuration. DNA cleavage experiments identified a pair of suitably oriented topoisomerase recognition sites, 90 bases from the centre of the cloned SFV terminal inverted repeat, which may mark the resolution site. These data suggest a resolution scheme in which branch migration of a Holliday junction through a site occupied by covalently bound topoisomerase molecules, could lead to telomere resolution.     

Characterization of the binding specificity of two anticruciform DNA monoclonal antibodies

Two monoclonal antibodies (2D3 and 4B4) have been raised against a stable cruciform DNA structure containing the 27-base pair palindrome of the SV40 origin of replication on one strand and an unrelated 26-base pair palindrome on the complementary strand (pRGM 21 x pRGM 29) and have been shown to recognize conformational determinants specific to cruciform DNA structures (Frappier, L., Price, G.B., Martin, R. G., and Zannis-Hadjopoulos, M. (1987) J. Mol. Biol. 193, 751-758). To define the region(s) of the cruciform that is recognized by these antibodies, we examined the ability of 2D3 and 4B4 to protect the single-stranded tips of the loops or the four-way junctions at the base of the stem of stable cruciform molecules against cleavage by mung bean nuclease or T7 endonuclease 3, respectively. Both antibodies were found to protect two of the four elbow-like structures at the base of the cruciform from T7 endonuclease 3 cleavage, but not the tips of the cruciform arms from mung bean nuclease cleavage. Also, predigestion of the cruciform with mung bean nuclease did not affect the binding of either antibody. In addition, 2D3 bound to a cruciform and a T-shaped structure involving the palindromic sequence at the cloning site of pUC7, which is completely unrelated in sequence to the palindrome of pRGM 21 x pRGM 29, and protected the base of these stem-loop structures against cleavage by T4 endonuclease VII. These results indicate that 2D3 and 4B4 bind at or near the base of the cruciform molecules and that, at least for 2D3, binding is independent of DNA sequence.     

Gene 3 endonuclease of bacteriophage T7 resolves conformationally branched structures in double-stranded DNA

Gene 3 endonuclease of bacteriophage T7 has been expressed from the cloned gene, purified, and characterized as to its activity on different DNA substrates. Besides its known strong preference for cutting single-stranded DNA rather than double-stranded DNA, the enzyme has a strong preference for cutting conformationally branched structures in double-stranded DNA, either X or Y-shaped branches. Three types of branched DNA substrates were used: relaxed circular DNAs containing large cruciform structures (a model for Holliday structures, presumed intermediates in genetic recombination); X-shaped molecules having a limited potential for branch migration, made from the cloned phage and bacterial arms of the lambda attachment site; and Y-shaped molecules, made by hybridizing molecules homologous except for a 2 X 21 base-pair palindrome in one of them. Gene 3 endonuclease cuts two opposing strands at or near the branchpoint to resolve these substrates into linear molecules, and does not cut the potentially single-stranded tips of the stem-and-loop structure generated from the palindrome. The position of the cleavage points on the equivalent arm of two X-shaped molecules, constructed from wild-type and mutant lambda attachment sites, show that the enzyme can cut at several different sites within or slightly 5' of the limited region of branch migration. The various activities of gene 3 endonuclease are consistent with the known role of this enzyme in genetic recombination, in maturation and packaging of T7 DNA, and in degradation of host DNA, and suggest that the enzyme recognizes a specific structural feature in DNA. Its cleavage specificity, ready availability, and ability to act at physiological pH and ionic conditions may make gene 3 endonuclease useful as a probe for specific DNA structures or for binding of proteins that alter DNA structure.     

Unequal excision of complementary strands is involved in the generation of palindromic repetitions of rho- mitochondrial DNA in yeast

In the rho- mutants of yeast, the mitochondrial genome is made up of a small segment excised from the wild-type mitochondrial DNA. The segment is repeated either in tandem or in palindrome to form a series of multimeric DNAs. We have asked how the palindromic organization arises. From several palindromic rho- mitochondrial DNAs, we have isolated the restriction fragments that contained the head-to-head or tail-to-tail junction of the repeating units, and have determined their nucleotide sequences. We found that the palindromes were not symmetrical right up to the junction points: at the junction, there was always an asymmetrical sequence of variable length. At both ends of this junction sequence, we found inverted oligonucleotide sequences that were variable in each mutant and that were present in the wild-type DNA. At the moment of excision, a single-strand cut seems to occur at each of these short inverted repeats, in such a way that the two complementary strands of the genome are cut unequally and the single-stranded overhangs become the junction sequences between the palindromic repeating units. This scheme may account for the complex structures of many rho- mitochondrial DNAs.