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.     

Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination

The characterization of plasmid-genomic DNA junctions following plant transformation has established links between DNA double-strand break repair (DSBR), illegitimate recombination and plasmid DNA integration. The limited information on plasmid-plasmid junctions in plants comes from the dicot species tobacco and Arabidopsis. We analyzed 12 representative transgenic rice lines, carrying a range of transforming plasmid rearrangements, which predominantly reflected microhomology mediated illegitimate recombination involving short complementary patches at the recombining ends. Direct end-ligation, in the absence of homology between the recombining molecules, occurred only rarely. Filler DNA was found at some of the junctions. Short, purine-rich tracts were present, either at the junction site or in the immediate flanking regions. Putative DNA topoisomerase I binding sites were clustered around the junctions. Although different regions of the transforming plasmid were involved in plasmid-plasmid recombination, we showed that a 19 bp palindromic sequence, including the TATA box of the CaMV 35S promoter, acted as a recombination hotspot. The purine-rich half of the palindromic sequence was specifically involved at the recombination junctions. This recombination hotspot is located within the 'highly recombinogenic' region of the full-length CaMV RNA that has been shown to promote viral recombination in dicot plants. Clustering of plasmid recombination events in this highly recombinogenic region, even in the absence of viral enzymes and other cis-acting elements proves that the plant cellular machinery alone is sufficient to recognize and act on these viral sequences. Our data also show the similarity between mechanisms underlying junction formation in dicot and monocot plants transformed using different procedures.     

The same mammalian replicon yields distinct recombination products in different cell lines.

We have observed previously that some chimeric replicons inclusive of a partly duplicated polyomavirus (Py) genome yield unit-length Py DNA (P155) at high frequency when transfected into normal or Py-transformed mouse cells. We demonstrate here that one such replicon generates either P155 or illegitimate recombination products in other mouse cells, transformed by simian virus 40. Use of the polymerase chain reaction indicates that each of the illegitimate products carried a different deletion, but that all deletions mapped within a rather well defined portion of the precursor replicon. Thus, these products were organized as if two hotspots for recombination existed in the Py late-coding region, one being located within or near one of the duplicated sequences characteristic of the chimeric replicon. Since this particular hotspot has already been shown to be involved in the generation of P155, the data reported here could indicate that a single recombination mechanism can yield either homologous (P155) or illegitimate products. How the DNA interacts with certain proteins, such as papovavirus large tumor antigen, could explain why one or the other type of product is formed.     

Hdf1, a yeast Ku-protein homologue, is involved in illegitimate recombination, but not in homologous recombination.

Hdf1 is the yeast homologue of the mammalian 70 kDa subunit of Ku-protein, which has DNA end-binding activity and is involved in DNA double-strand break repair and V(D)J recombination. To examine whether Hdf1 is involved in illegitimate recombination, we have measured the rate of deletion mutation caused by illegitimate recombination on a plasmid in an hdf1 disruptant. The hdf1 mutation reduced the rate of deletion formation by 20-fold, while it did not affect mitotic and meiotic homologous recombinations between two heteroalleles or homologous recombination between direct repeats. Hence Hdf1 participates in illegitimate recombination, but not in homologous recombination, in contrast to Rad52, Rad50, Mre11 and Xrs2, which are involved in both homologous and illegitimate recombination. The illegitimate recombination in the hdf1 disruptant took place between recombination sites that shared short regions of homology (1-4 bp), as was observed in the wild-type. Based on the DNA end-binding activity of Hdf1, we discuss models in which Hdf1 plays an important role in the late step of illegitimate recombination.     

Effect of the DNA topoisomerase II inhibitor VP-16 on illegitimate recombination in yeast chromosomes

Etoposide and teniposide, derivatives of podophyllotoxin, are inhibitors of DNA topoisomerase II and are potent anticancer agents. An adverse effect linked to the use of these drugs is the development of acute myeloid leukemia, a disorder usually associated with chromosomal translocation. To examine podophyllotoxin-induced DNA rearrangement, we developed an assay system to measure illegitimate recombination in Saccharomyces cerevisiae chromosomes. This approach uses juxtaposed CAN1-CYH2 negative selection markers that are introduced into the LEU2 locus, which is located on chromosome III, in a yeast strain carrying the mutated can1 and cyh2 genes. Upon formation of a deletion over the active CAN1-CYH2 genes, a cell becomes resistant to both canavanine and cycloheximide. To introduce drugs into the cell, we used a yeast strain carrying an ISE2 mutation, thereby making the cell drug-permeable. Here we show that treatment of cells with etoposide (VP-16) increases the rate of illegitimate recombination in yeast, indicating that VP-16 stimulates DNA topoisomerase-mediated illegitimate recombination. Structural analysis of the resulting recombinants indicate that most are formed by deletion mutations on chromosome III, which take place between short homologous regions of DNA. We propose a model for illegitimate recombination, in which VP-16 facilitates formation of a cleavable complex between DNA topoisomerase II and DNA, thus promoting DNA double-strand breakage with the resulting DNA ends joined by a non-homologous mechanism.     

Illegitimate recombination in Xenopus: characterization of end-joined junctions.

When linear DNAs are injected into Xenopus laevis eggs, they are converted into several different kinds of recombination products. Some molecules undergo homologous recombination by a resection-annealing mechanism; some ends are precisely ligated; and some ends are joined by illegitimate means. The homologous and illegitimate products are also generated in nuclear extracts from stage VI Xenopus oocytes. In order to gain insight into the mechanism(s) of illegitimate end joining, we amplified, cloned and sequenced a number of junctions from eggs and from oocyte extracts. The egg junctions fell into three categories: some with no homology at the join point that may have been produced by blunt-end ligation; some based on small, but significant homologies (5-10 bp); and some with matches of only 1 or 2 nucleotides at the joint. Junctions made in oocyte extracts were largely of the latter type. In the extracts, formation of illegitimate joints required the addition of all four deoxyribonucleoside triphosphates and was inhibited by aphidicolin. This indicates that this process involves DNA synthesis, and mechanisms incorporating this feature are considered. The spectrum of recombination products formed in Xenopus eggs is very reminiscent of those produced from DNA introduced into mammalian cells.     

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.     

Role of DNA ligase in the illegitimate recombination that generates lambdabio-transducing phages in Escherichia coli

We studied the role of DNA ligase in illegitimate recombination in Escherichia coli. A temperature-sensitive mutation in the lig gene reduced the frequency with which lambdabio-transducing phages were generated to 10-14% of that of wild type under UV irradiation. Reintroduction of the lig gene into this mutant restored the frequency of recombinant phage generation to that of wild type. Furthermore, overexpression of DNA ligase enhanced illegitimate recombination by 10-fold with or without UV irradiation. In addition, when DNA ligase was present in only limited amounts, UV-induced or spontaneous illegitimate recombination occurred exclusively at hotspot sites that have relatively long sequences of homology (9 or 13 bp). However, when DNA ligase was overexpressed, most of the illegitimate recombination took place at non-hotspot sites having only short sequences of homology (<4 bp). Thus, the level of ligase activity affects the frequency of illegitimate recombination, the length of sequence homology at the recombination sites, and the preference for recombination at hotspots, at least after UV irradiation. These observations support our hypothesis that the illegitimate recombination that generates lambdabio-transducing phages is mediated by the DNA break-and-join mechanism.     

Formation of heteroduplex DNA during mammalian intrachromosomal gene conversion.

We have studied intrachromosomal gene conversion in mouse Ltk- cells with a substrate designed to provide genetic evidence for heteroduplex DNA. Our recombination substrate consists of two defective chicken thymidine kinase genes arranged so as to favor the selection of gene conversion products. The gene intended to serve as the recipient in gene conversion differs from the donor sequence by virtue of a palindromic insertion that creates silent restriction site polymorphisms between the two genes. While selection for gene conversion at a XhoI linker insertion within the recipient gene results in coconversion of the nearby palindromic site in more than half of the convertants, 4% of convertant colonies show both parental and nonparental genotypes at the polymorphic site. We consider these mixed colonies to be the result of genotypic sectoring and interpret this sectoring to be a consequence of unrepaired heteroduplex DNA at the polymorphic palindromic site. DNA replication through the heteroduplex recombination intermediate generates genetically distinct daughter cells that comprise a single colony. We believe that the data provide the first compelling genetic evidence for the presence of heteroduplex DNA during chromosomal gene conversion in mammalian cells.     

RAD10, an excision repair gene of Saccharomyces cerevisiae, is involved in the RAD1 pathway of mitotic recombination.

The RAD10 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of UV-damaged DNA. We show that the RAD10 gene is also required for mitotic recombination. The rad10 delta mutation lowered the rate of intrachromosomal recombination of a his3 duplication in which one his3 allele has a deletion at the 3' end and the other his3 allele has a deletion at the 5' end (his3 delta 3' his3 delta 5'). The rate of formation of HIS3+ recombinants in the rad10 delta mutant was not affected by the rad1 delta mutation but decreased synergistically in the presence of the rad10 delta mutation in combination with the rad52 delta mutation. These observations indicate that the RAD1 and RAD10 genes function together in a mitotic recombination pathway that is distinct from the RAD52 recombination pathway. The rad10 delta mutation also lowered the efficiency of integration of linear DNA molecules and circular plasmids into homologous genomic sequences. We suggest that the RAD1 and RAD10 gene products act in recombination after the formation of the recombinogenic substrate. The rad1 delta and rad10 delta mutations did not affect meiotic intrachromosomal recombination of the his3 delta 3' his3 delta 5' duplication or mitotic and meiotic recombination of ade2 heteroalleles located on homologous chromosomes.     

An SMC-like protein is required for efficient homologous recombination in Arabidopsis.

In plants, the observed low frequency of gene targeting and intrachromosomal recombination contrasts markedly with the efficient extrachromosomal recombination of DNA. Thus, chromatin accessibility can have a major influence on the recombination frequency of chromosomal DNA in vivo. An Arabidopsis mutant hypersensitive to a range of DNA-damaging treatments (UV-C, X-rays, methyl methanesulfonate and mitomycin C) is also defective in somatic intrachromosomal homologous recombination. The wild-type gene encodes a protein closely related to the structural maintenance of chromosomes (SMC) family involved in structural changes in chromosomes. Although loss of SMC function is lethal in other eukaryotes, growth of the Arabidopsis mutant is normal in the absence of genotoxic treatments. This suggests a surprisingly specialized function for this protein in plants, and provides the first in vivo evidence for the involvement of an SMC protein in recombinational DNA repair. It is possible that SMC-like proteins in plants alleviate suppressive chromatin structure limiting homologous recombination in somatic cells.     

Chromosomal illegitimate recombination in mammalian cells is associated with intrinsically bent DNA elements.

Illegitimate recombination is the most frequent mechanism for chromosomal rearrangements in mammalian cells, yet little is known about this process. Most of the studies to date have looked at the sequences present at illegitimate junctions. These revealed the presence of recurrent DNA motifs, none of which was consistently found. We have undertaken to determine if intrinsic DNA structures such as bent DNA elements could be a major determinant in chromosomal illegitimate recombination. Using a two dimensional electrophoretic assay we found that eight out of eight junctions, resulting from various types of chromosomal rearrangements, had migration behaviour characteristic of DNA containing intrinsically bent DNA elements. In all cases, these occurred within one kilobase of the junctions, and in most cases could be found in both participating DNA segments. We also found that these bent DNA elements were present before the recombination event. When we analysed the frequency of intrinsically bent DNA elements in random chromosomal fragments, we found it to be about one per 11 kilobases. Thus these results suggest that bent DNA is associated with chromosomal illegitimate recombination.     

Stimulation of intrachromosomal homologous recombination in mammalian cells by an inhibitor of poly(ADP-ribosylation).

We determined the effect of 3-methoxybenzamide (3-MB), a competitive inhibitor of poly(ADP-ribose) polymerase (E.C. 2.4.2.30), on intrachromosomal homologous recombination in mouse Ltk- cells. We used a cell line that contained in its genome two defective Herpes thymidine kinase (tk) genes as closely linked direct repeats. Intrachromosomal homologous recombination events were monitored by selecting for tk-positive segregants that arose during propagation of the cells and recombination rates were determined by fluctuation analysis. We found that growth of cells in the continuous presence of 2mM 3-MB increased intrachromosomal recombination between 3 and 4-fold. Growth of cells in the presence of 2mM m-anisic acid, a non-inhibitory analog of 3-MB, had no effect on intrachromosomal recombination rates. Additionally, we found that 3-MB increased both gene conversions and crossovers to similar extents, adding to the evidence that these two types of intrachromosomal rearrangements share a common pathway. These findings contrast with our previous studies [Waldman, B.C. and Waldman, A.S. (1990) Nucleic Acids Res., 18, 5981-5988] in which we determined that 3-MB inhibits illegitimate recombination and has no effect on extrachromosomal homologous recombination in mouse Ltk- cells. An hypothesis is offered that explains the influence of 3-MB on different recombination pathways in mammalian cells in terms of the role that poly(ADP-ribosylation) plays in DNA break-repair.     

Direct-repeat analysis of chromatid interactions during intrachromosomal recombination in mouse cells.

Homologous intrachromosomal recombination between linked genes can involve interactions that are either intramolecular (intrachromatid) or intermolecular (sister chromatid). To assess the relative proportions of chromatid interactions, we report studies of intrachromosomal recombination in mouse L cells containing herpes simplex virus thymidine kinase genes in two alternative configurations of direct repeats. By comparing products of reciprocal exchanges between these two configurations, we conclude that the majority of interactions that give rise to crossover products involve unequally paired sister chromatids after DNA replication. Analyses of an additional class of crossover products that involve discontinuous associated gene conversion suggest that these recombination events involve a heteroduplex DNA intermediate.     

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.     

Molecular Analysis of λbio Transducing Phage Produced by Oxolinic Acid-Induced Illegitimate Recombination in Vivo

To study the mechanism of DNA gyrase-mediated illegitimate recombination in Escherichia coli, we examined the formation of λ Spi(-) phage during prophage induction. The frequency of Spi(-) phage was two to three orders of magnitude higher in the presence of oxolinic acid, an inhibitor of DNA gyrase A subunit, than in the absence of the drug, while it was very low in nalA(r) bacteria with the drug. RecA function is not required for the formation of these phages, indicating that this enhancement is not caused by the expression of SOS-controlled genes. Analyses of att region and recombination junctions of Spi(-) phages revealed that they have essentially the same structures as λbio transducing phages but are classified into two groups with respect to recombination sites. In the majority class of the transducing phages, there were not more than 3-bp homologies bewteen the parental E. coli bio and λ recombination sites. In the minority class of the transducing phages, on the other hand, 9-10-bp homologies were found between the parental recombination sites. These results suggested that oxolinic acid-induced illegitimate recombination takes place by two variants of a DNA gyrase-dependent mechanism.     

KIN17, a mouse nuclear protein, binds to bent DNA fragments that are found at illegitimate recombination junctions in mammalian cells

Illegitimate recombination is the dominant mechanism of recombination in mammalian somatic cells. It is responsible for most genome rearrangements such as translocations, deletions and integrations. Little is known as yet about the mechanism of illegitimate recombination and the enzymes involved. Recently, it has been shown that intrinsically bent DNA, also known as curved DNA, is present at chromosomal sites of illegitimate recombination events. Here we report that KIN17, a new mouse nuclear protein, binds to the curved DNA fragments found at illegitimate recombination sites.     

Induction of intrachromosomal homologous recombination in whole plants

The influence of different factors on frequencies of intrachromosomal homologous recombination in whole Arabidopsis thaliana and tobacco plants was analyzed using a disrupted β-glucuronidase marker gene. Recombination frequencies were enhanced severalfold by DNA damaging agents like UV-light or MMS (methyl methanesulfonate). Applying 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP)ribose polymerase (PARP), an enzyme that is postulated to be involved in DNA repair, enhanced homologous recombination frequencies strongly. These findings indicate that homologous recombination is involved in DNA repair and can (at least partially) compensate for other DNA repair pathways. Indications that recombination in plants can be induced by environmental stress factors that are not likely to be involved in DNA metabolism were also found; Arabidopsis plants growing in a medium containing 0.1 M NaCl exhibited elevated recombination frequencies. The possible general effects of ‘environmental’ challenges on genome flexibility are discussed.     

Targeted recombination in plants using Agrobacterium coincides with additional rearrangements at the target locus

The use of Agrobacterium for gene targeting in plants has been investigated. Leaf protoplasts of five transgenic tobacco lines, containing a T-DNA insertion with a defective npt-II gene at different positions in the plant genome, were transformed via Agrobacterium with a T-DNA containing a npt-II repair gene. After selection for kanamycin resistance and PCR analysis six recombinants were derived from four of the target lines. The recombination frequencies were similar for the different target lines with one recombinant from approximately 3×10⁵ transformants. Apparently gene targeting is more or less independent of the location of the target construct in the plant genome. Molecular analysis revealed that gene targeting had occurred in five of the six recombinant lines. However precise recombination had occurred in only one line, while in the other four lines restoration of the npt-II gene was accompanied by a deletion of part of the target locus. The sixth recombinant line showed restoration of the npt-II gene of the incoming T-DNA construct which was inserted in the plant genome at a position closely linked to the target locus. The different recombination products favour a model in which recombination is via gene conversion followed by reintegration of the synthesized DNA via homologous or illegitimate recombination rather than a reciprocal exchange of DNA between two cross-overs.     

The structures of integration sites in transgenic rice

Extensive genomic sequencing and sequence motif analysis have been conducted over the integration sites of two transgenic rice plants, #478 and #559, carrying the luciferase gene and/or hygromycin phosphotransferase gene. The transgenes reside in a region with inverted structure and a large duplication of rice genome over 2 kb. Integration was found at the AT-rich region and/or at the repetitive sequence region, including a SAR-like structure, retrotransposon and telomere repeats. The presence of a patch of sequence homology between plasmid and target DNA, and a small region of duplication involving the target DNA around the recombination site, implicated illegitimate recombination in the process of gene integration. Massive rearrangement of genomic DNA including deletion or translocation was also observed at the integration site and the flanking region of the transgene. The recognition sites of DNA topoisomerases I or II were observed in the rearranged sequences. Since only three junctions of transgenic rice were implicated in the illegitimate recombination and extensive rearrangement of the rice genome, rice protoplasts may be active in this process.