Different genome maintenance strategies in human and tobacco cells

In this work, genome maintenance strategies of organisms belonging to different kingdoms (animals versus plants) but of similar genome size were investigated using a novel, universal double-strand break (DSB) repair assay. Different plasmids linearised with KpnI, Acc65I or EcoRV yielding either 3' or 5' protruding or blunt DNA termini, respectively, were transfected into HeLa cells and Nicotiana plumbaginifolia protoplasts and assayed for the efficiency and fidelity of DSB repair. We show that the mechanism of break sealing is similar but that drastic differences are seen in the fidelity of repair: in HeLa cells, 50-55% DSBs were repaired precisely, compared to as little as 15-30% in tobacco cells. Moreover, the DSB repair in plants resulted in 30-40% longer deletions and significantly shorter insertions. Combined, these led to more than twofold larger net DNA loss in tobacco cells. Our observations point to possible differences in the strategies of DSB repair and genome maintenance in plants and animals.     

Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis

DNA double strand breaks (DSBs) occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ). We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum) by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1-20 bp) deletions occurred at a minority (25-45%) of repair junctions. Approximately ~1.5% of the observed repair events contained larger deletions (>20 bp) and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.     

The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution

The efficient repair of double-strand breaks (DSBs) in genomic DNA is important for the survival of all organisms. In recent years, basic mechanisms of DSB repair in somatic plant cells have been elucidated. DSBs are mainly repaired by non-homologous end-joining (NHEJ). The repair can be associated with deletions, but also insertions due to copying genomic sequences from elsewhere into the break. Species-specific differences of NHEJ have been reported and an inverse correlation of deletion size to genome size has been postulated, indicating that NHEJ might contribute significantly to evolution of genome size. DSB repair by homologous recombination (HR) might also influence genome organization. Whereas homology present in an allelic or an ectopic position is hardly used for repair, the use of homologous sequences in close proximity to the break is frequent. A 'single-strand annealing' mechanism that leads to sequence deletions between direct repeats is particularly efficient. This might explain the accumulation of single long terminal repeats of retroelements in cereal genomes. The conservative 'synthesis-dependent strand annealing' mechanism, resulting in conversions without crossovers is also prominent and seems to be significant for the evolution of tandemly arranged gene families such as resistance genes. Induction of DSBs could be used as a means for the controlled manipulation of plant genomes in an analogous way for the use of marker gene excision and site-specific integration.     

T-DNA integration in Arabidopsis chromosomes. Presence and origin of filler DNA sequences

To investigate the relationship between T-DNA integration and double-stranded break (DSB) repair in Arabidopsis, we studied 67 T-DNA/plant DNA junctions and 13 T-DNA/T-DNA junctions derived from transgenic plants. Three different types of T-DNA-associated joining could be distinguished. A minority of T-DNA/plant DNA junctions were joined by a simple ligation-like mechanism, resulting in a junction without microhomology or filler DNA insertions. For about one-half of all analyzed junctions, joining of the two ends occurred without insertion of filler sequences. For these junctions, microhomology was strikingly combined with deletions of the T-DNA ends. For the remaining plant DNA/T-DNA junctions, up to 51-bp-long filler sequences were present between plant DNA and T-DNA contiguous sequences. These filler segments are built from several short sequence motifs, identical to sequence blocks that occur in the T-DNA ends and/or the plant DNA close to the integration site. Mutual microhomologies among the sequence motifs that constitute a filler segment were frequently observed. When T-DNA integration and DSB repair were compared, the most conspicuous difference was the frequency and the structural organization of the filler insertions. In Arabidopsis, no filler insertions were found at DSB repair junctions. In maize (Zea mays) and tobacco (Nicotiana tabacum), DSB repair-associated filler was normally composed of simple, uninterrupted sequence blocks. Thus, although DSB repair and T-DNA integration are probably closely related, both mechanisms have some exclusive and specific characteristics.     

Differences in the processing of DNA ends in Arabidopsis thaliana and tobacco: possible implications for genome evolution

Surprising species-specific differences in non-homologous end-joining (NHEJ) of genomic double-strand breaks (DSBs) have been reported for the two dicotyledonous plants Arabidopsis thaliana and Nicotiana tabacum. In Arabidopsis deletions were, on average, larger than in tobacco and not associated with insertions. To establish the molecular basis of the phenomenon we analysed the fate of free DNA ends in both plant species by biolistic transformation of leaf tissue with linearized plasmid molecules. Southern blotting indicated that, irrespective of the nature of the ends (blunt, 5 or 3 overhangs), linearized full-length DNA molecules were, on average, more stable in tobacco than in Arabidopsis. The relative expression of a -glucuronidase gene encoded by the plasmid was similar in both plant species when the break was distant from the marker gene. However, if a DSB was introduced between the promoter and the open reading frame of the marker, transient expression was halved in Arabidopsisas compared to tobacco. These results indicate that free DNA ends are more stable in tobacco than in Arabidopsis, either due to lower DNA exonuclease activity or due to a better protection of DNA break ends or both. Exonucleolytic degradation of DNA ends might be a driving force in the evolution of genome size as the Arabidopsis genome is more than twenty times smaller than the tobacco genome.     

Global detection of DNA repair outcomes induced by CRISPR–Cas9

CRISPR–Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR–Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR–Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.     

Rapid sequence turnover at an intergenic locus in Drosophila

Closely related species of Drosophila tend to have similar genome sizes. The strong imbalance in favor of small deletions relative to insertions implies that the unconstrained DNA in Drosophila is unlikely to be passively inherited from even closely related ancestors, and yet most DNA in Drosophila genomes is intergenic and potentially unconstrained. In an attempt to investigate the maintenance of this intergenic DNA, we studied the evolution of an intergenic locus on the fourth chromosome of the Drosophila melanogaster genome. This 1.2-kb locus is marked by two distinct, large insertion events: a nuclear transposition of a mitochondrial sequence and a transposition of a nonautonomous DNA transposon DNAREP1_DM. Because we could trace the evolutionary histories of these sequences, we were able to reconstruct the length evolution of this region in some detail. We sequenced this locus in all four species of the D. melanogaster species complex: D. melanogaster, D. simulans, D. sechellia, and D. mauritiana. Although this locus is similar in size in these four species, less than 10% of the sequence from the most recent common ancestor remains in D. melanogaster and all of its sister species. This region appears to have increased in size through several distinct insertions in the ancestor of the D. melanogaster species complex and has been shrinking since the split of these lineages. In addition, we found no evidence suggesting that the size of this locus has been maintained over evolutionary time; these results are consistent with the model of a dynamic equilibrium between persistent DNA loss through small deletions and more sporadic DNA gain through less frequent but longer insertions. The apparent stability of genome size in Drosophila may belie very rapid sequence turnover at intergenic loci.     

Plant genome evolution: lessons from comparative genomics at the DNA level

Angiosperm genomes show tremendous variability in genome size and chromosome number. Nevertheless, comparative genetic mapping has revealed genome collinearity of closely related species. Sequence-based comparisons were used to assess the conservation of gene arrangements. Numerous small rearrangements, insertions/deletions, duplications, inversions and translocations have been detected. Importantly, comparative sequence analyses have unambiguously shown micro-collinearity of distantly related plant species. Duplications and subsequent gene loss have been identified as a particular important factor in the evolution of plant genomes.     

Mutational equilibrium model of genome size evolution

The paper describes a mutational equilibrium model of genome size evolution. This model is different from both adaptive and junk DNA models of genome size evolution in that it does not assume that genome size is maintained either by positive or stabilizing selection for the optimum genome size (as in adaptive theories) or by purifying selection against too much junk DNA (as in junk DNA theories). Instead the genome size is suggested to evolve until the loss of DNA through more frequent small deletions is equal to the rate of DNA gain through more frequent long insertions. The empirical basis for this theory is the finding of a strong correlation and of a clear power-function relationship between the rate of mutational DNA loss (per bp) through small deletions and genome size in animals. Genome size scales as a negative 1.3 power function of the deletion rate per nucleotide. Such a relationship is not predicted by either adaptive or junk DNA theories. However, if genome size is maintained at equilibrium by the balance of mutational forces, this empirilical relationship can be readily accommodated. Within this framework, this finding would imply that the rate of DNA gain through large insertions scales up a quarter-power function of genome size. On this view, as genome size grows, the rate of growth through large insertions is increasing as a quarter power function of genome size and the rate of DNA loss through small deletions increases linearly, until eventually, at the stable equilibrium genome size value, rates of growth and loss equal each other. The current data also suggest that the long-term variation is genome size in animals is brought about to a significant extent by changes in the intrinsic rates of DNA loss through small deletions. Both the origin of mutational biases and the adaptive consequences of such a mode of evolution of genome size are discussed.     

The shrunken genome of Arabidopsis thaliana

This paper examines macro and micro-level patterns of genome size evolution in the Brassicaceae. A phylogeny of 25 relatives of Arabidopsis thaliana was reconstructed using four molecular markers under both parsimony and Bayesian methods. Reconstruction of genome size (C value) evolution as a discrete character and as a continuous character was also performed. In addition, size dynamics in small chromosomal regions were assessed by comparing genomic clones generated for Arabidopsis lyrata and for Boechera stricta to the fully sequenced genome of A. thaliana. The results reveal a sevenfold variation in genome size among the taxa investigated and that the small genome size of A. thaliana is derived. Our results also indicate that the genome is free to increase or decrease in size across these evolutionary lineages without a directional bias. These changes are accomplished by insertions and deletions at both large and small-scales occurring mostly in intergenic regions, with repetitive sequences and transposable elements implicated in genome size increases. The focus upon taxa relatively closely related to the model organism A. thaliana, and the combination of complementary approaches, allows for unique insights into the processes driving genome size changes.     

Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells.

To analyze genomic changes resulting from double-strand break (DSB) repair, transgenic tobacco plants were obtained that carried in their genome a restriction site of the rare cutting endonuclease I-SceI within a negative selectable marker gene. After induction of DSB repair via Agrobacterium-mediated transient expression of I-SceI, plant cells were selected that carried a loss-of-function phenotype of the marker. Surprisingly, in addition to deletions, in a number of cases repair was associated with the insertion of unique and repetitive genomic sequences into the break. Thus, DSB repair offers a mechanism for spreading different kinds of sequences into new chromosomal positions. This may have evolutionary consequences particularly for plants, as genomic alterations occurring in meristem cells can be transferred to the next generation. Moreover, transfer DNA (T-DNA), carrying the open reading frame of I-SceI, was found in several cases to be integrated into the transgenic I-SceI site. This indicates that DSB repair also represents a pathway for the integration of T-DNA into the plant genome.     

Two unlinked double-strand breaks can induce reciprocal exchanges in plant genomes via homologous recombination and nonhomologous end joining

Using the rare-cutting endonuclease I-SceI we were able to demonstrate before that the repair of a single double-strand break (DSB) in a plant genome can be mutagenic due to insertions and deletions. However, during replication or due to irradiation several breaks might be induced simultaneously. To analyze the mutagenic potential of such a situation we established an experimental system in tobacco harboring two unlinked transgenes, each carrying an I-SceI site. After transient expression of I-SceI a kanamycin-resistance marker could be restored by joining two previously unlinked broken ends, either by homologous recombination (HR) or by nonhomologous end joining (NHEJ). Indeed, we were able to recover HR and NHEJ events with similar frequencies. Despite the fact that no selection was applied for joining the two other ends, the respective linkage could be detected in most cases tested, demonstrating that the respective exchanges were reciprocal. The frequencies obtained indicate that DSB-induced translocation is up to two orders of magnitude more frequent in somatic cells than ectopic gene conversion. Thus, DSB-induced reciprocal exchanges might play a significant role in plant genome evolution. The technique applied in this study may also be useful for the controlled exchange of unlinked sequences in plant genomes.     

Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms

In contrast to yeast RAD51, mammalian mRAD51 is an essential gene. Its role in double strand break (DSB) repair and its consequences on cell viability remain to be characterized precisely. Here, we used a hamster cell line carrying tandem repeat sequences with an I-SCE:I cleavage site. We characterized conservative recombination after I-SCE:I cleavage as gene conversion or intrachromatid crossing over associated with random reintegration of the excised reciprocal product. We identified two dominant-negative RAD51 forms that specifically inhibit conservative recombination: the yeast ScRAD51 or the yeast-mouse chimera SMRAD51. In contrast, the mouse MmRAD51 stimulates conservative recombination. None of these RAD51 forms affects non-conservative recombination or global DSB healing. Consistently, although resistance to gamma-rays remains unaffected, MmRAD51 stimulates whereas ScRAD51 or SMRAD51 prevents radiation-induced recombination. This suggests that mRAD51 does not significantly affect the global DSB repair efficiency but controls the classes of recombination events. Finally, both ScRAD51 and SMRAD51 drastically inhibit spontaneous recombination but not cell proliferation, showing that RAD51-dependent spontaneous and DSB-induced conservative recombination can be impaired significantly without affecting cell viability.     

Patching broken chromosomes with extranuclear cellular DNA

Chromosomal double-strand breaks (DSBs) can be repaired by either homology-dependent or homology-independent pathways. Using a novel intron-based genetic assay to identify rare homology-independent DNA rearrangements associated with repair of a chromosomal DSB in S. cerevisiae, we observed that approximately 20% of rearrangements involved endogenous DNA insertions at the break site. We have analyzed 37 inserts and find they fall into two distinct classes: Ty1 cDNA intermediates varying in length from 140 bp to 3.4 kb and short mitochondrial DNA fragments ranging in size from 33 bp to 219 bp. Several inserts consist of multiple noncontiguous mitochondrial DNA segments. These results demonstrate an ongoing mechanism for genome evolution through acquisition of organellar and mobile DNAs at DSB sites.     

Inter-species sequence comparison of Brachypodium reveals how transposon activity corrodes genome colinearity

Intergenic sequences evolve rapidly in plant genomes through a process known as genomic turnover. To investigate the influence of DNA transposons on genomic turnover, we compared 1 Mbp of orthologous genomic sequences from Brachypodium distachyon and Brachypodium sylvaticum. We found that B. distachyon and B. sylvaticum diverged approximately 1.7-2.0 million years ago. Of a total of 219 genes identified on the analyzed sequences, 211 were colinear. However, only 24 transposable elements of a total of 451 were orthologous (i.e. inserted in the common ancestor). We characterized in detail 59 insertions and 60 excisions of DNA transposons in one or other species, which altered 17% of the intergenic space. The DNA transposon excision sites showed complex and highly diagnostic sequence motifs for double-strand break (DSB) repair. DNA transposon excisions can lead to extensive deletions of hundreds of base pairs of flanking sequence if the DSB is repaired by 'single-strand annealing', or insertions of up to several hundred base pairs of 'filler DNA' if the DSB is repaired by 'synthesis-dependent strand annealing'. In some cases, DSBs were repaired by a combination of both methods. We present a model for the evolution of intergenic sequences in which repair of DSBs upon DNA transposon excision is a major factor in the rapid turnover and erosion of intergenic sequences.     

Pathways of DNA double-strand break repair and their impact on the prevention and formation of chromosomal aberrations

DNA double-strand breaks (DSB) are considered the critical primary lesion in the formation of chromosomal aberrations (CA). DSB occur spontaneously during the cell cycle and are induced by a variety of exogenous agents such as ionising radiation. To combat this potentially lethal damage, two related repair pathways, namely homologous recombination (HR) and non-homologous DNA end joining (NHEJ), have evolved, both of which are well conserved from bacteria to humans. Depending on the pathway used, the underlying mechanisms are capable of eliminating DSB without alterations to the original genomic sequence (error-free) but also may induce small scale mutations (base pair substitutions, deletions and/or insertions) and gross CA (error-prone). In this paper, we review the major pathways of DSB-repair, the proteins involved therein and their impact on the prevention of CA formation and carcinogenesis.     

Capture of DNA sequences at double-strand breaks in mammalian chromosomes

To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. To introduce a genomic DSB, cells were electroporated with a plasmid expressing endonuclease I-SceI, and clones that had lost tk function were selected. Among 253 clones analyzed, 78% displayed small deletions or insertions of several nucleotides at the DSB site. Surprisingly, approximately 8% of recovered mutations involved the capture of one or more DNA fragments. Among 21 clones that had captured DNA, 10 harbored a specific segment of the I-SceI expression plasmid mapping between two replication origins on the plasmid. Four clones had captured a long terminal repeat sequence from an intracisternal A particle (an endogenous retrovirus-like sequence) and one had captured what appears to be a cDNA copy of a moderately repetitive B2 sequence. Additional clones displayed segments of the tk gene and/or microsatellite sequences copied into the DSB. This first systematic study of DNA capture at DSBs in a mammalian genome suggests that DSB repair may play a considerable role in the evolution of eukaryotic genomes.     

Recombination drives vertebrate genome contraction

Selective and/or neutral processes may govern variation in DNA content and, ultimately, genome size. The observation in several organisms of a negative correlation between recombination rate and intron size could be compatible with a neutral model in which recombination is mutagenic for length changes. We used whole-genome data on small insertions and deletions within transposable elements from chicken and zebra finch to demonstrate clear links between recombination rate and a number of attributes of reduced DNA content. Recombination rate was negatively correlated with the length of introns, transposable elements, and intergenic spacer and with the rate of short insertions. Importantly, it was positively correlated with gene density, the rate of short deletions, the deletion bias, and the net change in sequence length. All these observations point at a pattern of more condensed genome structure in regions of high recombination. Based on the observed rates of small insertions and deletions and assuming that these rates are representative for the whole genome, we estimate that the genome of the most recent common ancestor of birds and lizards has lost nearly 20% of its DNA content up until the present. Expansion of transposable elements can counteract the effect of deletions in an equilibrium mutation model; however, since the activity of transposable elements has been low in the avian lineage, the deletion bias is likely to have had a significant effect on genome size evolution in dinosaurs and birds, contributing to the maintenance of a small genome. We also demonstrate that most of the observed correlations between recombination rate and genome contraction parameters are seen in the human genome, including for segregating indel polymorphisms. Our data are compatible with a neutral model in which recombination drives vertebrate genome size evolution and gives no direct support for a role of natural selection in this process.