Rapid, stabilizing palindrome rearrangements in somatic cells by the center-break mechanism

DNA palindromes are associated with rearrangement in a variety of organisms. A unique opportunity to examine the impact of a long palindrome in mammals is afforded by the Line 78 strain of mice. Previously it was found that the transgene in Line 78 is likely to be palindromic and that the symmetry of the transgene was responsible for a high level of germ line instability. Here we prove that Line 78 mice harbor a true 15.4-kb palindrome, and through the establishment of cell lines from Line 78 mice we have shown that the palindrome rearranges at the impressive rate of about 0.5% per population doubling. The rearrangements observed to arise from rapid palindrome modification are consistent with a center-break mechanism where double-strand breaks, created through hairpin nicking of an extruded cruciform, are imprecisely rejoined, thus introducing deletions at the palindrome center. Significantly, palindrome rearrangements in somatic tissue culture cells almost completely mirrored the structures generated in vivo in the mouse germ line. The close correspondence between germ line and somatic events indicates the possibility that center-break modification of palindromes is an important mechanism for preventing mutation in both contexts. Permanent cell lines carrying a verified palindrome provide an essential tool for future mechanistic analyses into the consequences of palindromy in the mammalian genome.     

AT-rich palindromes mediate the constitutional t(11;22) translocation

The constitutional t(11;22) translocation is the only known recurrent non-Robertsonian translocation in humans. Offspring are susceptible to der(22) syndrome, a severe congenital anomaly disorder caused by 3&rcolon;1 meiotic nondisjunction events. We previously localized the t(11;22) translocation breakpoint to a region on 22q11 within a low-copy repeat termed "LCR22" and within an AT-rich repeat on 11q23. The LCR22s are implicated in mediating different rearrangements on 22q11, leading to velocardiofacial syndrome/DiGeorge syndrome and cat-eye syndrome by homologous recombination mechanisms. The LCR22s contain AT-rich repetitive sequences, suggesting that such repeats may mediate the t(11;22) translocation. To determine the molecular basis of the translocation, we cloned and sequenced the t(11;22) breakpoint in the derivative 11 and 22 chromosomes in 13 unrelated carriers, including two de novo cases and der(22) syndrome offspring. We found that, in all cases examined, the reciprocal exchange occurred between similar AT-rich repeats on both chromosomes 11q23 and 22q11. To understand the mechanism, we examined the sequence of the breakpoint intervals in the derivative chromosomes and compared this with the deduced normal chromosomal sequence. A palindromic AT-rich sequence with a near-perfect hairpin could form, by intrastrand base-pairing, on the parental chromosomes. The sequence of the breakpoint junction in both derivatives indicates that the exchange events occurred at the center of symmetry of the palindromes, and this resulted in small, overlapping staggered deletions in this region among the different carriers. On the basis of previous studies performed in diverse organisms, we hypothesize that double-strand breaks may occur in the center of the palindrome, the tip of the putative hairpin, leading to illegitimate recombination events between similar AT-rich sequences on chromosomes 11 and 22, resulting in deletions and loss of the palindrome, which then could stabilize the DNA structure.     

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.     

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 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.     

An intramolecular recombination mechanism for the formation of the rRNA gene palindrome of Tetrahymena thermophila.

Large palindromic DNAs are found in a wide variety of eukaryotic cells. In Tetrahymena thermophila, a large palindrome is formed from a single rRNA gene (rDNA) during nuclear differentiation. We present evidence that a key step in the formation of the rDNA palindrome of T. thermophila involves homologous intramolecular recombination. Heteroduplex micronuclear rDNA molecules were constructed in vitro and microinjected into developing macronuclei, where they formed palindromes. Analysis of the resulting palindromes indicated that both strands of the microinjected rDNA are used to form the same palindrome. This study, together with a previous study (L. F. Yasuda and M.-C. Yao, Cell 67:505-516, 1991), is the first to define a molecular pathway of palindrome formation. The process is initiated by chromosome breakage at sites flanking the micronuclear rDNA. An intramolecular recombination reaction, guided by a pair of short inverted repeats located at the 5' end of the excised rDNA, covalently joins the two strands of micronuclear rDNA in a giant hairpin molecule. Bidirectional DNA replication converts the giant hairpin molecule to a palindrome. We suggest that the general features of this pathway are applicable to palindrome formation in other cell types.     

Palindromes and genomic stress fractures: bracing and repairing the damage

DNA palindromes are a source of instability in eukaryotic genomes but remain under-investigated because they are difficult to study. Nonetheless, progress in the last year or so has begun to form a coherent picture of how DNA palindromes cause damage in eukaryotes and how this damage is opposed by cellular mechanisms. In yeast, the features of double strand DNA interruptions that appear at palindromic sites in vivo suggest that a resolvase-type activity creates the fractures by attacking a palindrome after it extrudes into a cruciform structure. Induction of DNA breaks in this fashion could be deterred through a Center-Break palindrome revision process as investigated in detail in mice. The MRX/MRN likely plays a pivotal role in prevention of palindrome-induced genome damage in eukaryotes.     

A hotspot for novel amplification joints in a mosaic of Alu-like repeats and palindromic A + T-rich DNA.

We have identified, in the amplified domain of adenylate deaminase (AMPD) overproducing Chinese hamster fibroblasts, a 2.6 kb recombinogenic DNA region which is frequently involved in amplification-associated rearrangements. The nucleotide sequence reveals a mosaic organization of four Alu-equivalent repeats of the B1 and B2 families and eight long A + T-rich DNA segments. Part of this region is enriched with long imperfect palindromes. The center of one palindrome contains a putative topoisomerase I cleavage site and this site defines the position of a novel junction which was formed by illegitimate recombination with anther A + T-rich DNA sequence located far apart on the amplified DNA. These findings and their significance are discussed in the context of related data from other systems and in the light of current models for eukaryotic DNA recombination, replication and organization.     

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.     

Long palindromic sequences induce double-strand breaks during meiosis in yeast

Inverted-repeated or palindromic sequences have been found to occur in both prokaryotic and eukaryotic genomes. Such repeated sequences are usually short and present at several functionally important regions in the genome. However, long palindromic sequences are rare and are a major source of genomic instability. The palindrome-mediated genomic instability is believed to be due to cruciform or hairpin formation and subsequent cleavage of this structure by structure-specific nucleases. Here we present both genetic and physical evidence that long palindromic sequences (>50 bp) generate double-strand breaks (DSBs) at a high frequency during meiosis in the yeast Saccharomyces cerevisiae. The palindrome-mediated DSB formation depends on the primary sequence of the inverted repeat and the location and length of the repeated units. The DSB formation at the palindrome requires all of the gene products that are known to be responsible for DSB formation at the normal meiosis-specific sites. Since DSBs are initiators of nearly all meiotic recombination events, most of the palindrome-induced breaks appear to be repaired by homologous recombination. Our results suggest that short palindromic sequences are highly stable in vivo. In contrast, long palindromic sequences make the genome unstable by inducing DSBs and such sequences are usually removed from the genome by homologous recombination events.     

Myc oncogene-induced genomic instability: DNA palindromes in bursal lymphomagenesis

Genetic instability plays a key role in the formation of naturally occurring cancer. The formation of long DNA palindromes is a rate-limiting step in gene amplification, a common form of tumor-associated genetic instability. Genome-wide analysis of palindrome formation (GAPF) has detected both extensive palindrome formation and gene amplification, beginning early in tumorigenesis, in an experimental Myc-induced model tumor system in the chicken bursa of Fabricius. We determined that GAPF-detected palindromes are abundant and distributed nonrandomly throughout the genome of bursal lymphoma cells, frequently at preexisting short inverted repeats. By combining GAPF with chromatin immunoprecipitation (ChIP), we found a significant association between occupancy of gene-proximal Myc binding sites and the formation of palindromes. Numbers of palindromic loci correlate with increases in both levels of Myc over-expression and ChIP-detected occupancy of Myc binding sites in bursal cells. However, clonal analysis of chick DF-1 fibroblasts suggests that palindrome formation is a stochastic process occurring in individual cells at a small number of loci relative to much larger numbers of susceptible loci in the cell population and that the induction of palindromes is not involved in Myc-induced acute fibroblast transformation. GAPF-detected palindromes at the highly oncogenic bic/miR-155 locus in all of our preneoplastic and neoplastic bursal samples, but not in DNA from normal and other transformed cell types. This finding indicates very strong selection during bursal lymphomagenesis. Therefore, in addition to providing a platform for gene copy number change, palindromes may alter microRNA genes in a fashion that can contribute to cancer development.     

Comparison of physical and genetic properties of palindromic DNA sequences.

Some viable palindromic DNA sequences were found to cause an increase in the recovery of genetic recombinants. Although these palindromes contained no Chi sites, their presence in cis caused apparent recA+-dependent recombination to increase severalfold. This biological property did not correlate with the physical properties of the palindromes' extrusion of cruciform structures in vitro. Thus, two unrelated palindromes with similar effects on recombination in both Escherichia coli and Pseudomonas syringae displayed quite different kinetics of cruciform formation. In plasmids of native superhelical density, one palindrome underwent rapid cruciform formation at 55 degrees C, whereas the other did not form detectable cruciforms at any temperature. A shorter palindrome with similarly rapid kinetics of cruciform formation did not affect recombination detectably. The lack of a clear relationship between physical and genetic properties was also demonstrated in the case of longer, inviable palindromes. Here we found that the degree of asymmetry required in vivo to rescue a long palindrome from inviability far exceeded that required to kinetically prohibit cruciform extrusion in vitro.     

Palindromic sequences are associated with sites of DNA breakage during gene conversion.

Gene conversion is a recombinatorial mechanism which transfers genetic information from a donor into a recipient gene. A case of gene conversion between immunoglobulin VH region genes was analysed and palindromic sequences were found to be located near to the left recombinatorial breakpoint, which also is flanked by a direct repeat sequence. We performed a computer search for palindromes and direct repeats in the published sequences of eucaryotic genes which had been involved in gene conversion. In these sequences, the palindrome with the best or second best quality is located near to a breakpoint of recombination. A correlation of recombination breakpoints with direct repeats was not observed. This suggests that gene conversion is promoted by palindromic sequences.     

Palindromic sequences and A+T-rich DNA elements promote illegitimate recombination in Nicotiana tabacum.

Illegitimate recombination is the prevailing molecular mechanism for the integration of recombinant DNA into the genome of most eukaryotic systems and the generation of deletions by intrachromosomal recombination. We developed a ?selectable marker system to screen for intrachromosomal illegitimate recombination events in order to assess the sequence and structure-specific requirements for illegitimate recombination in tobacco. In 12 illegitimate recombination products analysed, we found that all deletion termini localise to sites of palindromic structures or to A+T-rich DNA elements. All deletion termini showed microhomologies of two to six nucleotides. In three plants, the recombination products contained filler-DNA or an inversion of an endogenous segment. Our data strongly suggest that illegitimate recombination in plants is mediated by a DNA synthesis-dependent process, and that this mechanism is promoted by DNA regions that can form palindromic structures or facilitate DNA unwinding.     

Fragile DNA Motifs Trigger Mutagenesis at Distant Chromosomal Loci in Saccharomyces cerevisiae

DNA sequences capable of adopting non-canonical secondary structures have been associated with gross-chromosomal rearrangements in humans and model organisms. Previously, we have shown that long inverted repeats that form hairpin and cruciform structures and triplex-forming GAA/TTC repeats induce the formation of double-strand breaks which trigger genome instability in yeast. In this study, we demonstrate that breakage at both inverted repeats and GAA/TTC repeats is augmented by defects in DNA replication. Increased fragility is associated with increased mutation levels in the reporter genes located as far as 8 kb from both sides of the repeats. The increase in mutations was dependent on the presence of inverted or GAA/TTC repeats and activity of the translesion polymerase Polζ. Mutagenesis induced by inverted repeats also required Sae2 which opens hairpin-capped breaks and initiates end resection. The amount of breakage at the repeats is an important determinant of mutations as a perfect palindromic sequence with inherently increased fragility was also found to elevate mutation rates even in replication-proficient strains. We hypothesize that the underlying mechanism for mutagenesis induced by fragile motifs involves the formation of long single-stranded regions in the broken chromosome, invasion of the undamaged sister chromatid for repair, and faulty DNA synthesis employing Polζ. These data demonstrate that repeat-mediated breaks pose a dual threat to eukaryotic genome integrity by inducing chromosomal aberrations as well as mutations in flanking genes.     

Formation of large palindromic DNA by homologous recombination of short inverted repeat sequences in Saccharomyces cerevisiae

Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes. The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms. Previously we have established that in Saccharomyces cerevisiae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence. In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation. Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences. We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation. We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation. In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1.     

Copy-choice illegitimate DNA recombination revisited.

Nearly precise excision of a transposon related to Tn10 from an Escherichia coli plasmid was used as a model to study illegitimate DNA recombination between short direct repeats. The excision was stimulated 100-1000 times by induction of plasmid single-stranded DNA synthesis and did not involve transfer of DNA from the parental to the progeny molecule. We conclude that it occurred by copy-choice DNA recombination, and propose that other events of recombination between short direct repeats might be a result of the same process.     

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.     

Role of recombination and replication fork restart in repeat instability

Eukaryotic genomes contain many repetitive DNA sequences that exhibit size instability. Some repeat elements have the added complication of being able to form secondary structures, such as hairpin loops, slipped DNA, triplex DNA or G-quadruplexes. Especially when repeat sequences are long, these DNA structures can form a significant impediment to DNA replication and repair, leading to DNA nicks, gaps, and breaks. In turn, repair or replication fork restart attempts within the repeat DNA can lead to addition or removal of repeat elements, which can sometimes lead to disease. One important DNA repair mechanism to maintain genomic integrity is recombination. Though early studies dismissed recombination as a mechanism driving repeat expansion and instability, recent results indicate that mitotic recombination is a key pathway operating within repetitive DNA. The action is two-fold: first, it is an important mechanism to repair nicks, gaps, breaks, or stalled forks to prevent chromosome fragility and protect cell health; second, recombination can cause repeat expansions or contractions, which can be deleterious. In this review, we summarize recent developments that illuminate the role of recombination in maintaining genome stability at DNA repeats.