Interchromatid and interhomolog recombination in Arabidopsis thaliana

Intermolecular recombination events were monitored in Arabidopsis thaliana lines using specially designed recombination traps consisting of tandem disrupted beta-glucuronidase or luciferase reporter genes in direct repeat orientation. Recombination frequencies (RFs) varied between the different lines, indicating possible position effects influencing intermolecular recombination processes. The RFs between sister chromatids and between homologous chromosomes were measured in plants either hemizygous or homozygous for a transgene locus. The RFs in homozygous plants exceeded those of hemizygous plants by a factor of >2, implying that in somatic plant cells both sister chromatid recombination and recombination between homologous chromosomes exist for recombinational DNA repair. In addition, different DNA-damaging agents stimulated recombination in homozygous and hemizygous plants to different extents in a manner dependent on the type of DNA damage and on the genomic region. The genetic and molecular analysis of recombination events showed that most of the somatic recombination events result from gene conversion, although a pop-out event has also been characterized.     

Somatic DNA recombination in a mouse genomic region, BC-1, in brain and non-brain tissue

The genomic region BC-1 (GenBank acc. No. AB075899) on mouse chromosome 16 has been reported as a genomic region undergoing somatic DNA recombination producing circular DNA and genomic deletion in brain during late embryogenesis. The present study shows that the BC-1 circular DNA production had already started on the 13th day of embryonic age, earlier than the previous observation that the circular DNA production started on the 15th through 17th embryonic day. The BC-1 deletion was also observed in the spleen and ocular lens. In situ hybridization analysis indicated that a human-homologous region in the BC-1 sequence was expressed in the lens at a perinatal period. These data suggest that the somatic DNA recombination in the BC-1 region is not restricted to brain tissue, and that the BC-1 DNA recombination relates to lens development.     

Somatic DNA recombination yielding circular DNA and deletion of a genomic region in embryonic brain

In this study, a mouse genomic region is identified that undergoes DNA rearrangement and yields circular DNA in brain during embryogenesis. External region-directed inverse polymerase chain reaction on circular DNA extracted from late embryonic brain tissue repeatedly detected DNA of this region containing recombination joints. Wide-range genomic PCR and digestion-circularization PCR analysis showed this region underwent recombination accompanied with deletion of intervening sequences, including the circularized regions. This region was mapped by fluorescence in situ hybridization to C1 on mouse chromosome 16, where no gene and no physiological DNA rearrangement had been identified. DNA sequence in the region has segmental homology to an orthologous region on human chromosome 3q.13. These observations demonstrated somatic DNA recombination yielding genomic deletions in brain during embryogenesis.     

Targeted modification of mammalian genomes

The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome. In most cell types, the frequency of homologous recombination is markedly lower than the frequency of random integration. Especially in somatic cells, homologous recombination is an extremely rare event. However, recent strategies involving the introduction of DNA double-strand breaks, triplex forming oligonucleotides or adeno-associated virus can increase the frequency of homologous recombination.

Site-specific recombination makes use of enzymes (recombinases, transposases, integrases), which catalyse DNA strand exchange between DNA molecules that have only limited sequence homology. The recognition sites of site-specific recombinases (e.g. Cre, Flp or ΦC31 integrase) are usually 30–50 bp. In contrast, retroviral integrases only require a specific dinucleotide sequence to insert the viral cDNA into the host genome. Depending on the individual enzyme, there are either innumerable or very few potential target sites for a particular integrase/recombinase in a mammalian genome. A number of strategies have been utilised successfully to alter the site-specificity of recombinases. Therefore, site-specific recombinases provide an attractive tool for the targeted modification of mammalian genomes.     

Effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on homologous recombination in somatic cells.

Sequencing studies have shown that in somatic cells alternating runs of purines and pyrimidines are frequently associated with recombination crossover points. To test whether such sequences actually promote recombination, we have examined the effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on a homologous recombination event. The parental molecule used in this study, pSVLD, is capable of generating wild-type simian virus 40 DNA via recombination across two 751-base-pair regions of homology and has been described previously (Miller et al., Proc. Natl. Acad. Sci. USA 81:7534-7538, 1984). Single inserts of either a poly[d(pGpT).d(pApC)] repeat or a poly[d(pCpG).d(pCpG)] repeat were positioned adjacent to one region of homology in such a way that the recombination product, wild-type simian virus 40 DNA, could be formed only by recombination within the homologies and not by recombination across the alternating purine-pyrimidine repeats. We have found that upon transfection of test DNAs into simian cells, a poly[d(pCpG).d(pCpG)] repeat enhanced homologous recombination 10- to 15-fold, whereas a poly[d(pGpT).d(pApC)] repeat had less effect. These results are discussed in terms of the features of these repeats that might be responsible for promoting homologous recombination.     

Rec A-independent homologous recombination induced by a putative fold-back tetraplex DNA

We have recently reported that a GC-rich palindromic repeat sequence presumably adopts a stable fold-back tetraplex DNA structure under supercoiling. To establish the biological significance of this structure, we inserted this sequence between two direct repeat sequences, separated by 200 bp, in a plasmid. We then investigated the effect of this sequence on homologous recombination events. Here we report that the putative fold-back DNA tetraplex structure induces homologous recombination between direct repeat sequences. Interestingly, this recombination event is independent of recA, a major driving force for homologous recombination. We think that the fold-back structure forces the repeat sequences to come into close proximity and therefore leads to strand exchange. Although triplex-induced recombination has been well documented, our results for the first time directly establish the potential of a tetraplex structure to induce recA-independent homologous recombination in vivo. This finding might have a significant implication for site-directed gene deletion in the context of the correction of genetic defects.     

Activation induced deaminase: the importance of being specific

Activation-induced deaminase (AID) is required for class switch recombination and somatic hypermutation in immunoglobulin genes. Although the preponderance of evidence suggests that AID functions by deaminating deoxycytidine in DNA, the question remains whether it can also deaminate cytidine in mRNA, as originally proposed based on its homology to RNA-editing enzymes. Recently, the biological relevance of assaying mammalian enzymes for DNA deaminase activity using Escherichia coli DNA as a reporter has been questioned, representing another round in the ongoing debate.     

Inferring processes underlying B-cell repertoire diversity

We quantify the VDJ recombination and somatic hypermutation processes in human B cells using probabilistic inference methods on high-throughput DNA sequence repertoires of human B-cell receptor heavy chains. Our analysis captures the statistical properties of the naive repertoire, first after its initial generation via VDJ recombination and then after selection for functionality. We also infer statistical properties of the somatic hypermutation machinery (exclusive of subsequent effects of selection). Our main results are the following: the B-cell repertoire is substantially more diverse than T-cell repertoires, owing to longer junctional insertions; sequences that pass initial selection are distinguished by having a higher probability of being generated in a VDJ recombination event; somatic hypermutations have a non-uniform distribution along the V gene that is well explained by an independent site model for the sequence context around the hypermutation site.     

Genetic mechanisms of tumor-specific loss of 11p DNA sequences in Wilms tumor.

Wilms tumor, a common childhood renal tumor, occurs in both a heritable and a nonheritable form. The heritable form may occasionally be attributed to a chromosome deletion at 11p13, and tumors from patients with normal constitutional chromosomes often show deletion or rearrangement of 11p13. It has been suggested that a germinal or somatic mutation may occur on one chromosome 11 and predispose to Wilms tumor and that a subsequent somatic genetic event on the normal homologue at 11p13 may permit tumor development. To study the frequency and mechanism of such tumor-specific genetic events, we have examined the karyotype and chromosome 11 genotype of normal and tumor tissues from 13 childhood renal tumor patients with different histologic tumor types and associated clinical conditions. Tumors of eight of the 12 Wilms tumor patients, including all viable tumors examined directly, show molecular evidence of loss of 11p DNA sequences by somatic recombination (four cases), chromosome loss (two cases), and recombination (two cases) or chromosome loss and duplication. One malignant rhabdoid tumor in a patient heterozygous for multiple 11p markers did not show any tumor-specific 11p alteration. These findings confirm the critical role of 11p sequences in Wilms tumor development and reveal that mitotic recombination may be the most frequent mechanism by which tumors develop.     

Agents that target telomerase and telomeres

Telomeres are guanine-rich regions that are located at the ends of chromosomes and are essential for preventing aberrant recombination and protecting against exonucleolytic DNA degradation. Telomeres are maintained by telomerase, an RNA-dependent DNA polymerase. Because telomerase is known to be expressed in tumor cells, which concurrently have short telomeres, and not in most somatic cells, which usually have long telomeres, telomerase and telomere structures have been recently proposed as attractive targets for the discovery of new anticancer agents. The most exciting current strategies are aimed at specifically designing new drugs that target telomerase or telomeres and new models have been formulated to study the biological effects of inhibitors of telomerase and telomeres both in vitro and in vivo.     

Germ line recombination may be primarily a manifestation of DNA repair processes.

Several kinds of DNA repair and reactivation processes have been found to occur by recombination in E. coli and its phages. Recombinational repair appears to be a major mechanism for overcoming lesions induced by various agents including ultraviolet light, X-rays, [³²P]-decay, nitrous acid and psoralen-plus-light. When the lesions caused by the above agents are not repaired by recombinational repair or other accurate repair modes they generally lead to lethality. However a minority of these unrepaired lesions lead to mutations through replication errors or error-prone repair. In higher organisms accumulation of unrepaired lethal lesions or mutations in somatic cells may cause aging. Perpetuation of a species depends on preventing accumulation of these types of defects in the germ line. It is proposed that the recombinational acts occurring during meiosis may largely reflect the recombinational removal or repair of DNA lesions to conserve the germ line. It is further suggested that the repair function of recombination may be as significant as the better studied function of recombination, the generation of diversity. The ubiquitous occurrence of recombination in the biological world implies that it arose very early in evolution. The possibility that recombination initially arose as a repair process is discussed.     

Chromosome breakage and recombination at fragile sites.

Chromosomal fragile sites are points on chromosomes that usually appear as nonstaining chromosome or chromatid gaps. It has frequently been suggested that fragile sites may be involved in chromosome breakage and recombination events. We and others have previously shown that fragile sites predispose to intrachromosomal recombination as measured by sister-chromatid exchanges. These findings suggested that fragile site expression often, if not always, is accompanied by DNA strand breakage. In the present report, fragile sites are shown to predispose to deletions and interchromosomal recombination. By use of somatic cell hybrids containing either human chromosome 3 or the fragile X chromosome, deletions and translocations were induced by FUdR or aphidicolin with breakpoints at the fragile sites Xq27 or 3p14.2 (FRA3B) or at points so close to the fragile sites as to be cytogenetically indistinguishable. Southern blot analysis of DNA from a panel of chromosome 3 deletion and translocation hybrids was then utilized to detect loss or retention of markers flanking FRA3B and to corroborate the cytogenetic evidence that the breakpoints were at this fragile site. One cell line with a reciprocal translocation between human chromosome 3 (with breakpoint at 3p14.2) and a hamster chromosome showed cytogenetically that the fragile site was expressed on both derivative chromosomes, supporting the hypothesis that the fragile site represents a repeated sequence. The approach described provides a means of generating specific rearrangements in somatic cell hybrids with a breakpoint at a fragile site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of Extra Chromosomal Recombination in Somatic Cells by Affecting the Ratio of Homologous Recombination (HR) to Non-Homologous End Joining (NHEJ)

Advancements in somatic cell gene targeting have been slow due to the finite lifespan of somatic cells and the overall inefficiency of homologous recombination. The rate of homologous recombination is determined by mechanisms of DNA repair, and by the balance between homologous recombination (HR) and non-homologous end joining (NHEJ). A plasmid-to-plasmid, extra chromosomal recombination system was used to study the effects of the manipulation of molecules involved in NHEJ (Mre11, Ku70/80, and p53) on HR/NHEJ ratios. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. While a mutant Mre11 and an anti-Ku aptamer did not significantly affect the rate of NHEJ or HR, transient expression of a p53 mutant increased overall HR/NHEJ by 2.5 fold. However, expression of the mutant p53 resulted in increased aneuploidy of the cultured cells. Additionally, we found no relationship between telomerase expression and changes in HR/NHEJ. In contrast, cell synchrony by thymidine incorporation did not induce chromosomal abnormalities, and increased the ratio of HR/NHEJ 5-fold by reducing the overall rate of NHEJ. Overall our results show that attempts at reducing NHEJ by use of Mre11 or anti-Ku aptamers were unsuccessful. Cell synchrony via thymidine incorporation, however, does increase the ratio of HR/NHEJ and this indicates that this approach may be of use to facilitate targeting in somatic cells by reducing the numbers of colonies that need to be analyzed before a HR is identified.     

Enhancement of extra chromosomal recombination in somatic cells by affecting the ratio of homologous recombination (HR) to non-homologous end joining (NHEJ)

Advancements in somatic cell gene targeting have been slow due to the finite lifespan of somatic cells and the overall inefficiency of homologous recombination. The rate of homologous recombination is determined by mechanisms of DNA repair, and by the balance between homologous recombination (HR) and non-homologous end joining (NHEJ). A plasmid-to-plasmid, extra chromosomal recombination system was used to study the effects of the manipulation of molecules involved in NHEJ (Mre11, Ku70/80, and p53) on HR/NHEJ ratios. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. While a mutant Mre11 and an anti-Ku aptamer did not significantly affect the rate of NHEJ or HR, transient expression of a p53 mutant increased overall HR/NHEJ by 2.5 fold. However, expression of the mutant p53 resulted in increased aneuploidy of the cultured cells. Additionally, we found no relationship between telomerase expression and changes in HR/NHEJ. In contrast, cell synchrony by thymidine incorporation did not induce chromosomal abnormalities, and increased the ratio of HR/NHEJ 5-fold by reducing the overall rate of NHEJ. Overall our results show that attempts at reducing NHEJ by use of Mre11 or anti-Ku aptamers were unsuccessful. Cell synchrony via thymidine incorporation, however, does increase the ratio of HR/NHEJ and this indicates that this approach may be of use to facilitate targeting in somatic cells by reducing the numbers of colonies that need to be analyzed before a HR is identified.     

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.     

Immunoglobulin class-switch recombination deficiencies

Immunoglobulin class-switch recombination deficiencies (Ig-CSR-Ds) are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect in question, the Ig-CSR-D may be combined with an impairment in somatic hypermutation (SHM). Some of the mechanisms underlying Ig-CSR and SHM have been described by studying natural mutants in humans. This approach has revealed that T cell-B cell interaction (resulting in CD40-mediated signaling), intrinsic B-cell mechanisms (activation-induced cytidine deaminase-induced DNA damage), and complex DNA repair machineries (including uracil-N-glycosylase and mismatch repair pathways) are all involved in class-switch recombination and SHM. However, several of the mechanisms required for full antibody maturation have yet to be defined. Elucidation of the molecular defects underlying the diverse set of Ig-CSR-Ds is essential for understanding Ig diversification and has prompted better definition of the clinical spectrum of diseases and the development of increasingly accurate diagnostic and therapeutic approaches.     

A hypervariable middle repetitive DNA sequence from citrus

The use of hypervariable sequences for DNA typing of plants is focussed on microsatellites and on amplification of regions defined by random (RAPD) or defined (AFLP) primers for PCR analysis of genomes. A hypervariable length of middle repetitive DNA has been isolated from citrus that contains no obvious hypervariable structures. The fingerprinting probe was shown to have an important commercial application in the separation of zygotic from nucellar progeny. A somatic variant of the sequence within one orange tree suggests that somatic variation in hypervariable markers may be a common event.     

Recombinogenic phenotype of human activation-induced cytosine deaminase

Class switch recombination, gene conversion, and somatic hypermutation that diversify rearranged Ig genes to produce various classes of high affinity Abs are dependent on the enzyme activation-induced cytosine deaminase (AID). Evidence suggests that somatic hypermutation is due to error-prone DNA repair that is initiated by AID-mediated deamination of cytosine in DNA, whereas the mechanism by which AID controls recombination remains to be elucidated. In this study, using a yeast model system, we have observed AID-dependent recombination. Expression of human AID in wild-type yeast is mutagenic for G-C to A-T transitions, and as expected, this mutagenesis is increased upon inactivation of uracil-DNA glycosylase. AID expression also strongly induces intragenic mitotic recombination, but only in a strain possessing uracil-DNA glycosylase. Thus, the initial step of base excision repair is required for AID-dependent recombination and is a branch point for either hypermutagenesis or recombination.     

The evolution of meiosis: recruitment and modification of somatic DNA-repair proteins

Several DNA-damage detection and repair mechanisms have evolved to repair double-strand breaks induced by mutagens. Later in evolutionary history, DNA single- and double-strand cuts made possible immune diversity by V(D)J recombination and recombination at meiosis. Such cuts are induced endogenously and are highly regulated and controlled. In meiosis, DNA cuts are essential for the initiation of homologous recombination, and for the formation of joint molecule and crossovers. Many proteins that function during somatic DNA-damage detection and repair are also active during homologous recombination. However, their meiotic functions may be altered from their somatic roles through localization, posttranslational modifications and/or interactions with meiosis-specific proteins. Presumably, somatic repair functions and meiotic recombination diverged during evolution, resulting in adaptations specific to sexual reproduction. (c) 2005 Wiley Periodicals, Inc.     

Crystal structure of a DNA Holliday junction.

DNA recombination is a universal biological event responsible both for the generation of genetic diversity and for the maintenance of genome integrity. A four-way DNA junction, also termed Holliday junction, is the key intermediate in nearly all recombination processes. This junction is the substrate of recombination enzymes that promote branch migration or catalyze its resolution. We have determined the crystal structure of a four-way DNA junction by multiwavelength anomalous diffraction, and refined it to 2.16 A resolution. The structure has two-fold symmetry, with pairwise stacking of the double-helical arms, which form two continuous B-DNA helices that run antiparallel, cross in a right-handed way, and contain two G-A mismatches. The exchanging backbones form a compact structure with strong van der Waals contacts and hydrogen bonds, implying that a conformational change must occur for the junction to branch-migrate or isomerize. At the branch point, two phosphate groups from one helix occupy the major groove of the other one, establishing sequence-specific hydrogen bonds. These interactions, together with different stacking energies and steric hindrances, explain the preference for a particular junction stacked conformer.