Nonrandom clusters of palindromes in herpesvirus genomes.

Palindromes are symmetrical words of DNA in the sense that they read exactly the same as their reverse complementary sequences. Representing the occurrences of palindromes in a DNA molecule as points on the unit interval, the scan statistics can be used to identify regions of unusually high concentration of palindromes. These regions have been associated with the replication origins on a few herpesviruses in previous studies. However, the use of scan statistics requires the assumption that the points representing the palindromes are independently and uniformly distributed on the unit interval. In this paper, we provide a mathematical basis for this assumption by showing that in randomly generated DNA sequences, the occurrences of palindromes can be approximated by a Poisson process. An easily computable upper bound on the Wasserstein distance between the palindrome process and the Poisson process is obtained. This bound is then used as a guide to choose an optimal palindrome length in the analysis of a collection of 16 herpesvirus genomes. Regions harboring significant palindrome clusters are identified and compared to known locations of replication origins. This analysis brings out a few interesting extensions of the scan statistics that can help formulate an algorithm for more accurate prediction of replication origins.     

Palindromes in proteins

Palindromes in DNA consist of nucleotides sequences that read the same from the 5'-end to the 3'-end, and its double helix is related by twofold axis. They occur in genomes of all organisms and have various functions. For example, restriction enzymes often recognize palindromic sequences of DNA. Palindromes in telomeres are crucial for initiation of replication. One can ask the questions, Do palindromes occur in protein, and if so, what function they play? We have searched the protein SWISSPROT database for palindromic sequences. A great number (26%) of different protein palindromes were found. One example of such protein is systemin, an 18-amino-acid-long peptide. It contains palindrome in its beta-sheet domain that interacts with palindromic fragment of DNA. The other palindrome containing protein is cellular human tumor suppressor p53. Oligonucleotide LTI-ITL has been observed in the crystal structure and is located close to a DNA recognizing domain. As the number of possible palindromic sequences of a given length is far much greater for proteins (20N) than for nucleic acids (4N), the study on their role seems to be an exciting challenge. Our results have clearly showed that palindromes are frequently occurring motives in proteins. Moreover, even very few examples that we have examined so far indicate the importance of further studies on protein palindromes.     

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.     

Palindromes in Proteins

Palindromes in DNA consist of nucleotides sequences that read the same from the 5′-end to the 3′-end, and its double helix is related by twofold axis. They occur in genomes of all organisms and have various functions. For example, restriction enzymes often recognize palindromic sequences of DNA. Palindromes in telomeres are crucial for initiation of replication. One can ask the questions, Do palindromes occur in protein, and if so, what function they play? We have searched the protein SWISSPROT database for palindromic sequences. A great number (26%) of different protein palindromes were found. One example of such protein is systemin, an 18-amino-acid-long peptide. It contains palindrome in its β-sheet domain that interacts with palindromic fragment of DNA. The other palindrome containing protein is cellular human tumor suppressor p53. Oligonucleotide LTIITL has been observed in the crystal structure and is located close to a DNA recognizing domain. As the number of possible palindromic sequences of a given length is far much greater for proteins (20^N) than for nucleic acids (4^N), the study on their role seems to be an exciting challenge. Our results have clearly showed that palindromes are frequently occurring motives in proteins. Moreover, even very few examples that we have examined so far indicate the importance of further studies on protein palindromes.     

Singular over-representation of an octameric palindrome, HIP1, in DNA from many cyanobacteria.

An octameric palindrome (5'-GCGATCGC-3') is abundant in cyanobacterial sequences within databases (GenBank/EMBL) and was designated HIP1 (highly iterated palindrome). The frequency of occurrence of all 256 octameric palindromes has now been determined in sub-databases revealing large and unique over-representation of HIP1 in cyanobacterial entries. DNA sequences from other bacteria were searched for any over-represented octameric palindromes analogous to HIP1. Only two sequences were identified, in the genomes of a thermophile and halophilic archaebacteria, although these were less abundant than HIP1 in cyanobacteria and relate to codon usage. To test the proposed widespread distribution of HIP1 in DNA from the cyanobacterium Synechococcus PCC 6301, randomly selected genomic clones were partly sequenced. HIP1 constituted 2.5% of the novel sequences, equivalent to a site on average once every 320 nucleotides. An oligonucleotide including HIP1 was also tested in PCR. Multiple products were obtained using template DNA from cyanobacterial strains in which HIP1 is abundant in known sequences, and some strains generated characteristic HIP-PCR banding patterns. However, analysis of DNA from one strain (not previously represented in databases) by random sequencing, HIP-PCR and Pvul digestion, confirms that not all cyanobacterial genomes are rich in HIP1.     

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.     

GAP-Seq: a method for identification of DNA palindromes

Background

Closely spaced long inverted repeats, also known as DNA palindromes, can undergo intrastrand annealing to form DNA hairpins. The ability to form these hairpins results in genome instability, difficulties in maintaining clones in Escherichia coli and major problems for most DNA sequencing approaches. Because of their role in genomic instability and gene amplification in some human cancers, it is important to develop systematic approaches to detect and characterize DNA palindromes.

Results

We developed a new protocol to identify palindromes that couples the S1 nuclease treated Cot0 DNA (GAPF) with high-throughput sequencing (GAP-Seq). Unlike earlier protocols, it does not involve restriction enzymatic digestion prior to DNA snap-back thereby preserving longer DNA sequences. It also indicates the location of the novel junction, which can then be recovered. Using MCF-7 breast cancer cell line as the proof-of-principle analysis, we have identified 35 palindrome candidates and physically characterized the top 5 candidates and their junctions. Because this protocol eliminates many of the false positives that plague earlier techniques, we have improved palindrome identification.

Conclusions

The GAP-Seq approach underscores the importance of developing new tools for identifying and characterizing palindromes, and provides a new strategy to systematically assess palindromes in genomes. It will be useful for studying human cancers and other diseases associated with palindromes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-394) contains supplementary material, which is available to authorized users.     

Compositional Bias is a Major Determinant of the Distribution Pattern and Abundance of Palindromes in Drosophila melanogaster

Palindromic sequences are important DNA motifs related to gene regulation, DNA replication and recombination, and thus, investigating the evolutionary forces shaping the distribution pattern and abundance of palindromes in the genome is substantially important. In this article, we analyzed the abundance of palindromes in the genome, and then explored the possible effects of several genomic factors on the palindrome distribution and abundance in Drosophila melanogaster. Our results show that the palindrome abundance in D. melanogaster deviates from random expectation and the uneven distribution of palindromes across the genome is associated with local GC content, recombination rate, and coding exon density. Our data suggest that base composition is the major determinant of the distribution pattern and abundance of palindromes and the correlation between palindrome density and recombination is a side-product of the effect of compositional bias on the palindrome abundance.     

回文序列诱导突变的机制及人类相关疾病

在原核和真核生物基因组中, 含有回文序列的区域高度可变且稳定性差, 主要原因是回文序列能形成发卡或十字形二级结构, 然后通过滑动错配、单链复性以及非同源末端连接(Non-homologous end joining, NHEJ)等机制导致缺失突变或染色体易位的发生。在人类基因组中, 回文序列较普遍存在于基因表达调控的重要作用元件中, 它诱导的缺失和易位突变还与男性不育、地中海贫血等多种疾病的发生、发展密切相关。文章综合近几年国内外相关文献, 初步阐释回文序列诱导突变的类型和可能机制, 及其与人类疾病的关系, 为进一步探讨回文序列在基因表达调控、基因突变及人类疾病中的作用及功能等相关研究提供参考。     

Size-dependent palindrome-induced intrachromosomal recombination in yeast

Palindromic and quasi-palindromic sequences are important DNA motifs found in various cis-acting genetic elements, but are also known to provoke different types of genetic alterations. The instability of such motifs is clearly size-related and depends on their potential to adopt secondary structures known as hairpins and cruciforms. Here we studied the influence of palindrome size on recombination between two directly repeated copies of the yeast CYC1 gene leading to the loss of the intervening sequence (“pop-out” recombination). We show that palindromes inserted either within one copy or between the two copies of the CYC1 gene become recombinogenic only when they attain a certain critical size and we estimate this critical size to be about 70 bp. With the longest palindrome used in this study (150 bp) we observed a more than 20-fold increase in the pop-out recombination. In the sae2/com1 mutant the palindrome-stimulated recombination was completely abolished. Suppression of palindrome recombinogenicity may be crucial for the maintenance of genetic stability in organisms containing a significant number of large palindromes in their genomes, like humans.     

Distribution of potential type II restriction sites (palindromes) in prokaryotes

Restriction-modification systems are used as a defensive mechanism against inappropriate invasion of foreign DNA. The recognition sequences for the common type II restriction enzymes and their corresponding methylases are usually palindromes. In this study, we identified the most over- and underrepresented words in DNA of four bacteria: Escherichia coli, Bacillus subtilis, Clostridium perfringens, and Pseudomonas aeruginosa. Using maximum order Markov chain analysis, we found that palindromic words were most often more underrepresented than their non-palindromic counterparts. No strict rule for the intragenic palindrome content could be derived, but for three of the bacteria there was a weak correlation between codon usage bias and palindrome content. A clear drop in palindrome counts was observed in the Shine-Dalgarno region for B. subtilis and C. perfringens, but not in E. coli or P. aeruginosa. It was also shown that palindromes in eubacteria and archaebacteria seem to occur slightly more infrequently than expected on the basis of the genomic GC-content, but some exceptions to this principle exist.     

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.     

Intrastrand annealing leads to the formation of a large DNA palindrome and determines the boundaries of genomic amplification in human cancer

Amplification of large chromosomal regions (gene amplification) is a common somatic alteration in human cancer cells and often is associated with advanced disease. A critical event initiating gene amplification is a DNA double-strand break (DSB), which is immediately followed by the formation of a large DNA palindrome. Large DNA palindromes are frequent and nonrandomly distributed in the genomes of cancer cells and facilitate a further increase in copy number. Although the importance of the formation of large DNA palindromes as a very early event in gene amplification is widely recognized, it is not known how a DSB is resolved to form a large DNA palindrome and whether any local DNA structure determines the location of large DNA palindromes. We show here that intrastrand annealing following a DNA double-strand break leads to the formation of large DNA palindromes and that DNA inverted repeats in the genome determine the efficiency of this event. Furthermore, in human Colo320DM cancer cells, a DNA inverted repeat in the genome marks the border between amplified and nonamplified DNA. Therefore, an early step of gene amplification is a regulated process that is facilitated by DNA inverted repeats in the genome.     

The rabbit C family of short, interspersed repeats. Nucleotide sequence determination and transcriptional analysis

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication (rep-) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted (ori-) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori- mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori- mutant also complemented replication of AAV rep- mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.     

Large DNA palindromes as a common form of structural chromosome aberrations in human cancers

Breakage-fusion-bridge cycles contribute to chromosome aberrations and generate large DNA palindromes that facilitate oncogene amplification in cancer cells. At the molecular level, large DNA palindrome formation is initiated by chromosome breaks, and genomic architecture such as short inverted repeat sequences facilitates this process in mammalian cells. However, the prevalence of DNA palindromes in cancer cells is currently unknown. To determine the prevalence of DNA palindromes in human cancer cells, we have developed a new microarray-based approach called Genome-wide Analysis of Palindrome Formation (GAPF, Tanaka et al., Nat Genet 2005; 37: 320-7). This approach is based on a relatively simple and efficient method to purify "snap-back DNA" from large DNA palindromes by intramolecular base-pairing, followed by elimination of single-stranded DNA by nuclease S1. Comparison of Genome-wide Analysis of Palindrome Formation profiles between cancer and normal cells using microarray can identify genome-wide distributions of somatic palindromes. Using a human cDNA microarray, we have shown that DNA palindromes occur frequently in human cancer cell lines and primary medulloblastomas. Significant overlap of the loci containing DNA palindromes between Colo320DM and MCF7 cancer cell lines suggests regions in the genome susceptible to chromosome breaks and palindrome formation. A subset of loci containing palindromes is associated with gene amplification in Colo320DM, indicating that the location of palindromes in the cancer genome serves as a structural platform that supports subsequent gene amplification.     

Effect of magnesium on cruciform extrusion in supercoiled DNA.

Recently, it was reported that Mg2+greatly facilitates cruciform extrusion in the short palindromes of supercoiled DNA, thereby allowing the formation of cruciform structures in vivo. Because of the potential biological importance of this phenomenon, we undertook a broader study of the effect of Mg2+on a cruciform extrusion in supercoiled DNA. The method of two-dimensional gel electrophoresis was used to detect the cruciform extrusion both in the absence and in the presence of these ions. Our results show that Mg2+shifts the cruciform extrusion in the d(CCC(AT)16GGG) palindrome to a higher, rather than to a lower level of supercoiling. In order to study possible sequence-specific properties of the short palindromes for which the unusual cruciform extrusion in the presence Mg2+was reported, we constructed a plasmid with a longer palindromic region. This region bears the same sequences in the hairpin loops and four-arm junction as the short palindrome, except that the short stems of the hairpins are extended. The extension allowed us to overcome the limitation of our experimental approach which cannot be used for very short palindromes. Our results show that Mg2+also shifts the cruciform extrusion in this palindrome to a higher level of supercoiling. These data suggest that cruciform extrusion in the short palindromes at low supercoiling is highly improbable. We performed a thermodynamic analysis of the effect of Mg2+on cruciform extrusion. The treatment accounted for the effect of Mg2+on both free energy of supercoiling and the free energy of cruciform structure per se. Our analysis showed that although the level of supercoiling required for the cruciform extrusion is not reduced by Mg2+, the ions reduce the free energy of the cruciform structure.     

A paucity of palindromes in φX174

The nucleotide sequence of the bacteriophage φX174 contains surprisingly few restriction endonuclease recognition sites. The observed frequency of those sites which consist of a six-nucleotide palindromic sequence would occur by chance with a probability of less than 8 × 10^?5. The genome of φX174 does not contain the four-nucleotide palindromic recognition site for the enzyme MboI and this finding has a probability of only 1·7 × 10^?9. A further analysis of the nucleotide sequence revealed that there is a marked scarcity of palindromic sequences with a length of four or six nucleotides while all other palindromes occur with a frequency close to that dictated by chance. A preliminary analysis of the genomes of other DNA viruses indicated that this palindrome avoidance is associated with single-stranded viruses only. The reasons for the paucity of these short even-numbered palindromes remains to be determined.     

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.     

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.