Stabilization of Microsatellite Sequences by Variant Repeats in the Yeast Saccharomyces Cerevisiae

We examined the effect of a single variant repeat on the stability of a 51-base pair (bp) microsatellite (poly GT). We found that the insertion stabilizes the microsatellite about fivefold in wild-type strains. The stabilizing effect of the variant base was also observed in strains with mutations in the DNA mismatch repair genes pms1, msh2 and msh3, indicating that this effect does not require a functional DNA mismatch repair system. Most of the microsatellite alterations in the pms1, msh2 and msh3 strains were additions or deletions of single GT repeats, but about half of the alterations in the wild-type and msh6 strains were large (>8 bp) deletions or additions.     

Instability of simple sequence DNA in Saccharomyces cerevisiae.

All eukaryotic genomes thus far examined contain simple sequence repeats. A particularly common simple sequence in many organisms (including humans) consists of tracts of alternating GT residues on one strand. Allelic poly(GT) tracts are often of different lengths in different individuals, indicating that they are likely to be unstable. We examined the instability of poly(GT) and poly(G) tracts in the yeast Saccharomyces cerevisiae. We found that these tracts were dramatically unstable, altering length at a minimal rate of 10(-4) events per division. Most of the changes involved one or two repeat unit additions or deletions, although one alteration involved an interaction with the yeast telomeres.     

Characterization of a multisubunit human protein which selectively binds single stranded d(GA)n and d(GT)n sequence repeats in DNA.

A protein which selectively binds d(GA)n and d(GT)n sequence repeats in single stranded DNA has been identified in human fibroblasts. This protein, designated PGB, has been purified at least 500-fold by ammonium sulfate precipitation followed by DEAE-Sepharose column chromatography and affinity chromatography in a column of d(GA)-Sepharose. Electrophoretic mobility shift assays revealed that the PGB protein bound most avidly d(GA)n and d(GT)n tracts of n > 5. It also bound other G-rich DNA sequence repeats, including dGn tracts, with lower affinities. It did not manifest significant binding affinities to single stranded M13 DNA, or to the homopolynucleotides poly dA, poly dC and poly dT, or to various DNA sequence repeats which do not contain G residues, such as d(A-C)n and d(TC)n. It did not bind double stranded d(T-C)n.d(GA)n tracts or other double stranded DNA sequences. In glycerol gradient centrifugation assays the d(GA)n- and the d(GT)n-binding activities cosedimented as a homogeneous protein species having an S20,w = 9.4 +/- 0.7 and an estimated native molecular weight of 190,000 +/- 7,000. UV crosslinking assays revealed that the protein contains 33.6 +/- 2.1 kd subunits which bind d(GA)n and d(GT)n sequences. However, SDS-polyacrylamide gel electrophoresis of the purified protein followed by silver staining indicated that it may also contain other subunits that do not contact the DNA. It is proposed that binding of the PGB protein to single stranded d(GA)n or d(GT)n tracts in double stranded topologically restricted DNA may stimulate strand separation and formation of triple helices or other unusual DNA structures.     

Two distinct models account for short and long deletions within sequence repeats in Escherichia coli.

In Escherichia coli, (GpC)n sequences cloned into plasmid DNA molecules are deletion-prone with the occurrence of both short (<2 bp) and long (>2 bp) deletion events. These repetitive tracts can be stabilized by interrupting the strict monotony of the repetition with a variant dinucleotide sequence. The stabilization of short deletion events that is mediated by the variant sequence is completely lost in E. coli mismatch repair-deficient strains. In contrast, this repair pathway has no influence on the frequency of occurrence of long deletion events, even in sequences containing the variant repeat. These results lead us to propose two distinct models to account for short and long deletions within repetitive sequences in E. coli. Furthermore, this study reveals that the deletions occur preferentially at the end of the repeat sequence that is distal with respect to the origin of replication.     

The primary structure of the mitochondrial genome of Saccharomyces cerevisiae--a review

We have collated and compiled all the available primary structure data on the mitochondrial genome of Saccharomyces cerevisiae. Data concern 78,500 bp, namely 92% of the 'long' genomes; they are derived from several laboratory strains. Interstrain differences belong to three classes: a small number of large deletions/additions, mainly concerning introns; a large number of small (10-150 bp) deletions/additions located in the intergenic sequences; 1-3 bp deletions/additions and point mutations; the interstrain sequence divergence due to the latter, is of the order of 2% for the strains compared; this low value is, however, an overestimate because of sequence mistakes.     

Instability of a Plasmid-Borne Inverted Repeat in Saccharomyces Cerevisiae

Inverted repeated DNA sequences are common in both prokaryotes and eukaryotes. We found that a plasmid-borne 94 base-pair inverted repeat (a perfect palindrome of 47 bp) containing a poly GT sequence is unstable in S. cerevisiae, with a minimal deletion frequency of about 10(-4)/mitotic division. Ten independent deletions had identical end points. Sequence analysis indicated that all deletions were the result of a DNA polymerase slippage event (or a recombination event) involving a 5-bp repeat (5' CGACG 3') that flanked the inverted repeat. The deletion rate and the types of deletions were unaffected by the rad52 mutation. Strains with the pms1 mutation had a 10-fold elevated frequency of instability of the inverted repeat. The types of sequence alterations observed in the pms1 background, however, were different than those seen in either the wild-type or rad52 genetic backgrounds.     

DNA sequence analysis of X-ray-induced deletions at the white locus of Drosophila melanogaster

We have determined the nucleotide sequence of 5 X-ray-induced white mutants containing small rearrangements. Comparison with wild-type sequences showed deletions in the coding region ranging in size between 6 bp and 29 bp. These small deletions are distributed non-randomly over the white locus. Two mutants contain the same 29-bp deletion, while the other 3 deletions are clustered. All 5 deletions have occurred between 2 and 3 bp repeats. One of the repeats is preserved in the novel junction formed by the deletion. Our results suggest that recombinational processes may be involved in the generation of X-ray-induced deletions.     

Targeting DNA polymerase to DNA double-strand breaks reduces DNA deletion size and increases templated insertions generated by CRISPR/Cas9

Most insertions or deletions generated by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) endonucleases are short (<25 bp), but unpredictable on-target long DNA deletions (>500 bp) can be observed. The possibility of generating long on-target DNA deletions poses safety risks to somatic genome editing and makes the outcomes of genome editing less predictable. Methods for generating refined mutations are desirable but currently unavailable. Here, we show that fusing Escherichia coli DNA polymerase I or the Klenow fragment to Cas9 greatly increases the frequencies of 1-bp deletions and decreases >1-bp deletions or insertions. Importantly, doing so also greatly decreases the generation of long deletions, including those >2 kb. In addition, templated insertions (the insertion of the nucleotide 4 nt upstream of the protospacer adjacent motif) were increased relative to other insertions. Counteracting DNA resection was one of the mechanisms perturbing deletion sizes. Targeting DNA polymerase to double-strand breaks did not increase off-targets or base substitution rates around the cleavage sites, yet increased editing efficiency in primary cells. Our strategy makes it possible to generate refined DNA mutations for improved safety without sacrificing efficiency of genome editing.     

Structure-dependent recombination hot spot activity of GAA.TTC sequences from intron 1 of the Friedreich's ataxia gene

The recombinational properties of long GAA.TTC repeating sequences were analyzed in Escherichia coli to gain further insights into the molecular mechanisms of the genetic instability of this tract as possibly related to the etiology of Friedreich's ataxia. Intramolecular and intermolecular recombination studies showed that the frequency of recombination between the GAA.TTC tracts was as much as 15 times higher than the non-repeating control sequences. Homologous, intramolecular recombination between GAA.TTC tracts and GAAGGA.TCCTTC repeats also occurred with a very high frequency (approximately 0.8%). Biochemical analyses of the recombination products demonstrated the expansions and deletions of the GAA.TTC repeats. These results, together with our previous studies on the CTG.CAG sequences, suggest that the recombinational hot spot characteristics may be a common feature of all triplet repeat sequences. Unexpectedly, we found that the recombination properties of the GAA.TTC tracts were unique, compared with CTG.CAG repeats, because they depended on the DNA secondary structure polymorphism. Increasing the length of the GAA.TTC repeats decreased the intramolecular recombination frequency between these tracts. Also, a correlation was found between the propensity of the GAA.TTC tracts to adopt the sticky DNA conformation and the inhibition of intramolecular recombination. The use of novobiocin to modulate the intracellular DNA topology, i.e. the lowering of the negative superhelical density, repressed the formation of the sticky DNA structure, thereby restoring the expected positive correlation between the length of the GAA.TTC tracts and the frequency of intramolecular recombination. Hence, our results demonstrate that sticky DNA exists and functions in E. coli.     

The rates and patterns of deletions in the human factor IX gene.

Deletions are commonly observed in genes with either segments of highly homologous sequences or excessive gene length. However, in the factor IX gene and in most genes, deletions (of > or = 21 bp) are uncommon. We have analyzed DNA from 290 families with hemophilia B (203 independent mutations) and have found 12 deletions > 20 bp. Eleven of these are > 2 kb (range > 3-163 kb), and one is 1.1 kb. The junctions of the four deletions that are completely contained within the factor IX gene have been determined. A novel mutation occurred in patient HB128: the data suggest that a 26.8-kb deletion occurred between two segments of alternating purines and pyrimidines and that a 2.3-kb sense strand segment derived from the deleted region was inserted. For our sample of 203 independent mutations, we estimate the "baseline" rates of deletional mutation per base pair per generation as a function of size. The rate for large (> 2 kb) deletions is exceedingly low. For every mutational event in which a given base is at the junction of a large deletion, there are an estimated 58 microdeletions (< 20 bp) and 985 single-base substitutions at that base. Analysis of the nine reported deletion junctions in the factor IX gene literature reveals that (i) five are associated with inversions, orphan sequences, or sense strand insertions; (ii) four are simple deletions that display an excess of short direct repeats at their junctions; (iii) there is no dramatic clustering of junctions within the gene; and (iv) with the exception of alternating purines and pyrimidines, deletion junctions are not preferentially associated with repetitive DNA.     

Direct and Inverted Repeats Elicit Genetic Instability by Both Exploiting and Eluding DNA Double-Strand Break Repair Systems in Mycobacteria

Repetitive DNA sequences with the potential to form alternative DNA conformations, such as slipped structures and cruciforms, can induce genetic instability by promoting replication errors and by serving as a substrate for DNA repair proteins, which may lead to DNA double-strand breaks (DSBs). However, the contribution of each of the DSB repair pathways, homologous recombination (HR), non-homologous end-joining (NHEJ) and single-strand annealing (SSA), to this sort of genetic instability is not fully understood. Herein, we assessed the genome-wide distribution of repetitive DNA sequences in the Mycobacterium smegmatis, Mycobacterium tuberculosis and Escherichia coli genomes, and determined the types and frequencies of genetic instability induced by direct and inverted repeats, both in the presence and in the absence of HR, NHEJ, and SSA. All three genomes are strongly enriched in direct repeats and modestly enriched in inverted repeats. When using chromosomally integrated constructs in M. smegmatis, direct repeats induced the perfect deletion of their intervening sequences ∼1,000-fold above background. Absence of HR further enhanced these perfect deletions, whereas absence of NHEJ or SSA had no influence, suggesting compromised replication fidelity. In contrast, inverted repeats induced perfect deletions only in the absence of SSA. Both direct and inverted repeats stimulated excision of the constructs from the attB integration sites independently of HR, NHEJ, or SSA. With episomal constructs, direct and inverted repeats triggered DNA instability by activating nucleolytic activity, and absence of the DSB repair pathways (in the order NHEJ>HR>SSA) exacerbated this instability. Thus, direct and inverted repeats may elicit genetic instability in mycobacteria by 1) directly interfering with replication fidelity, 2) stimulating the three main DSB repair pathways, and 3) enticing L5 site-specific recombination.     

Familial transmission of the FMR1 CGG repeat.

To better define the nature of FMR1 CGG-repeat expansions, changes in allele sizes for 191 families with fragile X and for 33 families with gray-zone repeats (40-60) were analyzed. Expansion of the fragile X chromosome to the full mutation was seen in 13.4% of offspring from premutation mothers with 56-59 repeats, 20.6% of those with 60-69 repeats, 57.8% of those with 70-79 repeats, 72.9% of those with 80-89 repeats, and 97.3% of those with 90-199 repeats. For premutation fathers, the majority (62%) of their daughters had a larger repeat number, while a few had either a smaller (22%) or the same (16%) repeat number, compared with their fathers' sizes. However, daughters with a smaller repeat number were observed only if their fathers had > or = 80 repeats. Fifteen (39.5%) of 38 such daughters carried a smaller repeat than did their fathers. We observed that a similar repeat number was inherited more often than expected by chance, among the members of a sibship segregating fragile X. This familial clustering, observed in the offspring of both males and females with a premutation, implies there may be an additional factor, independent of parental repeat size, that influences CGG-repeat instability. Instability in gray-zone allele transmissions was observed in 25% of alleles with 50-60 CGGs but in <8% of those with 40-49 CGGs. Examination of gray-zone allele organization revealed that long tracts of pure CGGs (>34) are not always unstably transmitted. These results raise new questions regarding the familial factors that may determine transmission expansions.     

Control of GT repeat stability in Schizosaccharomyces pombe by mismatch repair factors

The mismatch repair (MMR) system ensures genome integrity by removing mispaired and unpaired bases that originate during replication. A major source of mutational changes is strand slippage in repetitive DNA sequences without concomitant repair. We established a genetic assay that allows measuring the stability of GT repeats in the ade6 gene of Schizosaccharomyces pombe. In repair-proficient strains most of the repeat variations were insertions, with addition of two nucleotides being the most frequent event. GT repeats were highly destabilized in strains defective in msh2 or pms1. In these backgrounds, mainly 2-bp insertions and 2-bp deletions occurred. Surprisingly, essentially the same high mutation rate was found with mutants defective in msh6. In contrast, a defect in swi4 (a homologue of Msh3) caused only slight effects, and instability was not further increased in msh6 swi4 double mutants. Also inactivation of exo1, which encodes an exonuclease that has an MMR-dependent function in repair of base-base mismatches, caused only slightly increased repeat instability. We conclude that Msh2, Msh6, and Pms1 have an important role in preventing tract length variations in dinucleotide repeats. Exo1 and Swi4 have a minor function, which is at least partially independent of MMR.     

High somatic instability of a microsatellite locus in a clonal tree,<Emphasis Type="Italic"> Robinia pseudoacacia</Emphasis>

Robinia pseudoacacia L. is a clonal tree species. To investigate a mutation within eight microsatellite loci of R. pseudoacacia, we analyzed DNA samples obtained from different leaf samples within each ramet, leaves from ramets within the genet, and seeds. Of the eight loci, locus Rops15 (AG motif) displayed hypermutability. The mutation rates of Rops15 within each ramet, among ramets within the genet, and offspring were 6.27% (ranging from 0 to 31.1%), 6.11% (from 0 to 25.0%) and 3.78% (from 0 to 10.9%), respectively. The mutation rate increased with allele size (13–71 repeat units). The mutation patterns observed in Rops15 were distinctive in two ways. First, there was a significant bias toward additions over deletions, and both addition and deletion of single repeats were dominant at alleles with lengths less than 232 bp (63 repeats). Second, for the longest allele of 248 bp (71 repeats), the number of losses was higher than the number of gains. These observations suggest that the mutation patterns of microsatellites in R. pseudoacacia may follow a generalized stepwise mutation model, and that the tendency of long alleles to mutate to shorter lengths acts to prevent infinite growth. Finally, the observation of somatic hypermutability at locus Rops15 highlights the need for caution when using highly polymorphic microsatellites for population genetic structure and paternity analysis in tree species.Communicated by H.F. Linskens     

Inverted DNA repeats: a source of eukaryotic genomic instability.

While inverted DNA repeats are generally acknowledged to be an important source of genetic instability in prokaryotes, relatively little is known about their effects in eukaryotes. Using bacterial transposon Tn5 and its derivatives, we demonstrate that long inverted repeats also cause genetic instability leading to deletion in the yeast Saccharomyces cerevisiae. Furthermore, they induce homologous recombination. Replication plays a major role in the deletion formation. Deletions are stimulated by a mutation in the DNA polymerase delta gene (pol3). The majority of deletions result from imprecise excision between small (4- to 6-bp) repeats in a polar fashion, and they often generate quasipalindrome structures that subsequently may be highly unstable. Breakpoints are clustered near the ends of the long inverted repeats (< 150 bp). The repeats have both intra- and interchromosomal effects in that they also create hot spots for mitotic interchromosomal recombination. Intragenic recombination is 4 to 18 times more frequent for heteroalleles in which one of the two mutations is due to the insertion of a long inverted repeat, compared with other pairs of heteroalleles in which neither mutation has a long repeat. We propose that both deletion and recombination are the result of altered replication at the basal part of the stem formed by the inverted repeats.     

Instability of long inverted repeats within mouse transgenes.

Various sequences in the mammalian genomes are unstable. One class of sequence arrangement is long inverted repeats, which are known to be unstable in bacteria and yeast. While in mammals some evidence suggests that short inverted repeats (<10 bp long) may show instability, nothing is known about the stability of long inverted repeats. Here we describe two unrelated multicopy transgenes in the mouse (loci 109 and OX1-5), each of which contains a long inverted repeat that shows substantial mitotic instability. This instability also occurs in the germline so that mutant transgenes appear within pedigrees at a high frequency. The mutation processes acting at these two inverted repeats are complex and can involve insertion or deletion, and can result in stabilization of the transgene. At transgene 109 mutational events range from very small rearrangements at the centre of the inverted repeat to complete transgene deletion. In addition we show that the rates of mutation at the inverted repeat of transgene OX1-5 can vary between the male and female germlines and between inbred strains of mice, suggesting the possibility of a genetic analysis to identify loci that modulate inverted repeat instability.     

Genome-wide mutagenesis resulting from topoisomerase 1-processing of unrepaired ribonucleotides in DNA

Ribonucleotides are the most common non-canonical nucleotides incorporated into DNA during replication, and their processing leads to mutations and genome instability. Yeast mutation reporter systems demonstrate that 2–5 base pair deletions (Δ2–5bp) in repetitive DNA are a signature of unrepaired ribonucleotides, and that these events are initiated by topoisomerase 1 (Top1) cleavage. However, a detailed understanding of the frequency and locations of ribonucleotide-dependent mutational events across the genome has been lacking. Here we present the results of genome-wide mutational analysis of yeast strains deficient in Ribonucleotide Excision Repair (RER). We identified mutations that accumulated over thousands of generations in strains expressing either wild-type or variant replicase alleles (M644G Pol ε, L612M Pol δ, L868M Pol α) that confer increased ribonucleotide incorporation into DNA. Using a custom-designed mutation-calling pipeline called muver (for mutationes verificatae), we observe a number of surprising mutagenic features. This includes a 24-fold preferential elevation of AG and AC relative to AT dinucleotide deletions in the absence of RER, suggesting specificity for Top1-initiated deletion mutagenesis. Moreover, deletion rates in di- and trinucleotide repeat tracts increase exponentially with tract length. Consistent with biochemical and reporter gene mutational analysis, these deletions are no longer observed upon deletion of TOP1. Taken together, results from these analyses demonstrate the global impact of genomic ribonucleotide processing by Top1 on genome integrity.     

Lead acetate and arsenic trioxide induce instability of microsatellites at three different fragile sites (6q21, 9q32-9q33 and 15p14) within the genome of the rat

Human exposure to metals is of increasing concern due to the well-documented toxic and carcinogenic effects of metals and metal compounds, and the rising environmental levels due to industrial processes and pollution. It has been reported that metals can be genotoxic by several modes of action including generation of reactive oxygen species and inhibition of DNA repair. The aim of this study was to evaluate microsatellite instability (MSI) in three microsatellite loci (D6mit3, D9mit2 and D15Mgh1) located within three common fragile sites in the genome of the laboratory rat (6q21, 9q32-9q33 and 15p14) exposed to acute and chronic doses of a metal salt (lead acetate trihydrate) and a metalloid oxide (arsenic trioxide). In the acute and sub-chronic studies with the two compounds, MSI was observed in the three loci as deletions or additions of microsatellite repeats. The percentages of MSI were 36.4% and 42.1% for lead acetate and arsenic trioxide, respectively. Results of the present work indicate that the microsatellites located within fragile sites provide a convenient assay system to detect changes in DNA sequences resulting from exposure to genotoxic agents.