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1.
Creation of a chloroplast microsatellite reporter for detection of replication slippage in Chlamydomonas reinhardtii
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Microsatellites are composed of short tandem direct repeats; deletions or duplications of those repeats through the process of replication slippage result in microsatellite instability relative to other genomic loci. Variation in repeat number occurs so frequently that microsatellites can be used for genotyping and forensic analysis. However, an accurate assessment of the rates of change can be difficult because the presence of many repeats makes it difficult to determine whether changes have occurred through single or multiple events. The current study was undertaken to experimentally assess the rates of replication slippage that occur in vivo in the chloroplast DNA of Chlamydomonas reinhardtii. A reporter construct was created in which a stretch of AAAG repeats was inserted into a functional gene to allow changes to be observed when they occurred at the synthetic microsatellite. Restoration of the reading frame occurred through replication slippage in 15 of every million viable cells. Since only one-third of the potential insertion/deletion events would restore the reading frame, the frequency of change could be deduced to be 4.5 x 10(-5). Analysis of the slippage events showed that template slippage was the primary event, resulting in deletions rather than duplications. These findings contrasted with events observed in Escherichia coli during maintenance of the plasmid, where duplications were the rule. 相似文献
2.
Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA 总被引:12,自引:0,他引:12
Y. Ogihara K. Isono T. Kojima A. Endo M. Hanaoka T. Shiina T. Terachi S. Utsugi M. Murata N. Mori S. Takumi K. Ikeo T. Gojobori R. Murai K. Murai Y. Matsuoka Y. Ohnishi H. Tajiri K. Tsunewaki 《Molecular genetics and genomics : MGG》2002,266(5):740-746
Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize. 相似文献
3.
Proliferation of direct repeats near the Oenothera chloroplast DNA origin of replication 总被引:1,自引:0,他引:1
The spacer between the 16S and 23S rRNA genes of the chloroplast DNA has
been implicated as an origin of replication in several species of plants.
In the evening primrose, Oenothera, this site was found to vary greatly in
size, with plastid genomes (plastomes) being readily distinguished. To
determine whether plastome "strength" in transmission could be correlated
with variation at oriB, the 16S rRNA-trnI spacer was sequenced from five
plastomes. The size variation was found to be due to differential
amplification (and deletion) of combinations of sequences belonging to
seven families of direct repeats. From these comparisons, one short series
of direct repeats and one region capable of forming a hairpin structure
were identified as candidates for the factor that could be responsible for
the differences between strong and weak plastome types. Ample sequence
variation allowed phylogenetic inferences to be made about the
relationships among the plastomes. Phylogenetic trees also could be
constructed for most of the families of direct repeats. The amplifications
and deletions of repeats that account for the size variation at oriB are
proposed to have occurred through extensive replication slippage at this
site.
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4.
Wan-Ling Chiu Ellen M. Johnson Sara A. Kaplan Kimberly Blasko Mary B. Sokalski Ruth Wolfson Barbara B. Sears 《Molecular & general genetics : MGG》1990,221(1):59-64
Summary
Oenothera plants homozygous for a recessive allele at the plastome mutator (pm) locus show non-Mendelian mutation frequencies that are 1000-fold higher than spontaneous levels. Chloroplast DNA (cpDNA) was isolated from nine mutants and two green isolates of the plastome mutator line. cpDNA restriction patterns were compared to cpDNA from a representative of the progenitor Johansen strain, and cpDNAs from all eleven plastome mutator lines show changes of fragment mobility due to deletion events at five discrete regions of the plastome. Most of the mutants have cpDNA restriction patterns identical to that of one of the green isolates from the plastome mutator line, and therefore, most of the differences in fragment length are probably not responsible for the mutant phenotypes. In contrast to the plastome mutator line, cpDNA from several populations of a closely related wild-type Oenothera species have few restriction fragment length polymorphisms. This suggests that both mutation frequencies and site-specific cpDNA deletions are elevated in the plastome mutator line, and implicates a defect in the cpDNA repair or replication machinery. 相似文献
5.
6.
Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes. 总被引:6,自引:5,他引:1
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H T Tran N P Degtyareva N N Koloteva A Sugino H Masumoto D A Gordenin M A Resnick 《Molecular and cellular biology》1995,15(10):5607-5617
Small direct repeats, which are frequent in all genomes, are a potential source of genome instability. To study the occurrence and genetic control of repeat-associated deletions, we developed a system in the yeast Saccharomyces cerevisiae that was based on small direct repeats separated by either random sequences or inverted repeats. Deletions were examined in the LYS2 gene, using a set of 31- to 156-bp inserts that included inserts with no apparent potential for secondary structure as well as two quasipalindromes. All inserts were flanked by 6- to 9-bp direct repeats of LYS2 sequence, providing an opportunity for Lys+ reversion via precise excision. Reversions could arise by extended deletions involving either direct repeats or random sequences and by -1-or +2-bp frameshift mutations. The deletion breakpoints were always associated with short (3- to 9-bp) perfect or imperfect direct repeats. Compared with the POL+ strain, deletions between small direct repeats were increased as much as 100-fold, and the spectrum was changed in a temperature-sensitive DNA polymerase delta pol3-t mutant, suggesting a role for replication. The type of deletion depended on orientation relative to the origin of replication. On the basis of these results, we propose (i) that extended deletions between small repeats arise by replication slippage and (ii) that the deletions occur primarily in either the leading or lagging strand. The RAD50 and RAD52 genes, which are required for the recombinational repair of many kinds of DNA double-strand breaks, appeared to be required also for the production of up to 90% of the deletions arising between separated repeats in the pol3-t mutant, suggesting a newly identified role for these genes in genome stability and possibly replication. 相似文献
7.
Analysis of repeat-mediated deletions in the mitochondrial genome of Saccharomyces cerevisiae
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Mitochondrial DNA deletions and point mutations accumulate in an age-dependent manner in mammals. The mitochondrial genome in aging humans often displays a 4977-bp deletion flanked by short direct repeats. Additionally, direct repeats flank two-thirds of the reported mitochondrial DNA deletions. The mechanism by which these deletions arise is unknown, but direct-repeat-mediated deletions involving polymerase slippage, homologous recombination, and nonhomologous end joining have been proposed. We have developed a genetic reporter to measure the rate at which direct-repeat-mediated deletions arise in the mitochondrial genome of Saccharomyces cerevisiae. Here we analyze the effect of repeat size and heterology between repeats on the rate of deletions. We find that the dependence on homology for repeat-mediated deletions is linear down to 33 bp. Heterology between repeats does not affect the deletion rate substantially. Analysis of recombination products suggests that the deletions are produced by at least two different pathways, one that generates only deletions and one that appears to generate both deletions and reciprocal products of recombination. We discuss how this reporter may be used to identify the proteins in yeast that have an impact on the generation of direct-repeat-mediated deletions. 相似文献
8.
Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis. We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA. We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat. Resumption of DNA replication then completes the slippage process. 相似文献
9.
Deletions of muscle mitochondrial DNA in mitochondrial myopathies: sequence analysis and possible mechanisms. 总被引:9,自引:2,他引:7
Forty per cent of patients with mitochondrial myopathies, a diverse group of multisystem diseases predominantly affecting skeletal muscle and the brain, have large deletions of a proportion of muscle mitochondrial DNA (mt DNA). These appeared to be identical in 13 of 28 cases, contained within the region 8286-13595 bp. Analysis of the deletion junction in two cases showed a 13 nucleotide sequence which occurred in the normal genome as a direct repeat flanking the region deleted in the mutant mt DNAs. Mt DNA deletions may arise from recombination or slippage between short sequence repeats during replication. 相似文献
10.
11.
Ismail Iraqui Yasmina Chekkal Nada Jmari Violena Pietrobon Karine Fréon Audrey Costes Sarah A. E. Lambert 《PLoS genetics》2012,8(10)
Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination. 相似文献
12.
Mutational specificity of the dnaE173 mutator associated with a defect in the catalytic subunit of DNA polymerase III of Escherichia coli. 总被引:4,自引:0,他引:4
We developed a system to examine forward mutations that occurred in the rpsL gene of Escherichia coli placed on a multicopy plasmid. Using this system we determined the mutational specificity for a dnaE173 mutator strain in which the editing function of DNA polymerase III is impeded. The frequency of rpsL- mutations increased 32,000-fold, due to the dnaE173 mutator, and 87 independent rpsL- mutations in the mutator strain were analyzed by DNA sequencing, together with 100 mutants recovered from dnaE+ strain, as the control. While half the number of mutations that occurred in the wild-type strain were caused by insertion elements, no such mutations were recovered from the mutator strain. A novel class of mutation, named "sequence substitution" was present in mutants raised in the dnaE173 strain; seven sequence substitutions induced in the mutator strain occurred at six sites, and all were located in quasipalindromic sequences, carrying the GTG or CAC sequence at one or both endpoints. While other types of mutation were found in both strains, single-base frameshifts were the most frequent events in the mutator strain. Thus, the mutator effect on this class of mutation was 175,000-fold. A total of 95% of the single-base frameshifts in the mutator strain were additions, most of which occurred at runs of A or C bases so as to increase the number of identical residues. Base substitutions, the frequency of which was enhanced 25,000-fold by the mutator effect, occurred primarily at several hotspots in the mutator strain, whereas those induced in the wild-type strain were more randomly distributed throughout the rpsL sequence. The dnaE173 mutator also increased the frequency of duplications 28,000-fold. Of the three duplications recovered from the mutator strain, one was a simple duplication, the region of which was flanked by direct repeats. The other duplications were complex, one half part of which was in the inverted orientation of a region containing two sets of inverted repeats. The same duplications were also recovered from the wild-type strain. The present data suggest that dnaE173 is a novel class of mutator that sharply induces sequence-directed mutagenesis, yielding high frequencies of single base frameshifts, duplications with inversions, sequence substitutions and base substitutions at hotspots. 相似文献
13.
Different mechanisms inferred from sequences of human mitochondrial DNA deletions in ocular myopathies. 总被引:7,自引:1,他引:6
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F Degoul I Nelson S Amselem N Romero B Obermaier-Kusser G Ponsot C Marsac P Lestienne 《Nucleic acids research》1991,19(3):493-496
We have sequenced the deletion borders of the muscle mitochondrial DNA from 24 patients with heteroplasmic deletions. The length of these deletions varies from 2.310 bp to 8.476 bp and spans from position 5.786 to 15.925 of the human mitochondrial genome preserving the heavy chain and light chain origins of replication. 12 cases are common deletions identical to the mutation already described by other workers and characterized by 13 bp repeats at the deletion boundaries, one of these repeats being retained during the deletion process. The other cases (10 out of 12) have shown deletions which have not been previously described. All these deletions are located in the H strand DNA region which is potentially single stranded during mitochondrial DNA replication. In two cases, the retained Adenosine from repeat closed to the heavy strand origin of replication would indicate slippage mispairing. Furthermore in one patient two mt DNA molecules have been cloned and their sequences showed the difference of four nucleotides in the breakpoint of the deletion, possibly dued to slippage mispairing. Taken together our results suggest that deletions occur either by slippage mispairing or by internal recombination at the direct repeat level. They also suggest that different mechanisms account for the deletions since similarly located deletions may display different motives at the boundaries including the absence of any direct repeat. 相似文献
14.
Nonhomologous RNA recombination during negative-strand synthesis of flock house virus RNA. 总被引:2,自引:13,他引:2
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During sequential replicative passages of viral RNA from the nodavirus flock house virus, spontaneous deletion of RNA sequences occurred frequently. Families of deleted RNA molecules were derived from both segments of the bipartite viral genome and found to contain single, double, or triple deletions. These deletions were attributed to template switching by the flock house virus RNA replicase, resulting in recombination between distant sequences and excision of the intervening nucleotides. From sequence analysis of the recombination junctions, we concluded that the process of template switching was influenced by both the primary sequence and the secondary structure of the RNA and that it occurred predominantly during synthesis of RNA negative strands. 相似文献
15.
The mechanism of disease-associated (CTG)*(CAG) expansion may involve DNA replication slippage, replication direction, Okazaki fragment processing, recombination, or repair. A length-dependent bias for expansions is observed in humans affected by a trinucleotide repeat-associated disease. We developed an assay to test the effect of replication direction on (CTG)*(CAG) instabilities incurred during in vitro (SV40) DNA replication mediated by human cell extracts. This system recapitulates the bias for expansions observed in humans. Replication by HeLa cell extracts generated expansions and deletions that depended upon repeat tract length and the direction of replication. Templates with 79 repeats yielded predominantly expansions (CAG as lagging strand template) or predominantly deletions (CTG as lagging strand template). Templates containing 17 repeats were stable. Thus, replication direction determined the type of mutation. These results provide new insights into the orientation of replication effect upon repeat stability. This system will be useful in determining the contribution of specific human proteins to (CTG)*(CAG) expansions. 相似文献
16.
Analysis of immunoglobulin Sgamma3 recombination breakpoints by PCR: implications for the mechanism of isotype switching. 总被引:2,自引:0,他引:2
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The molecular mechanism of immunoglobulin switch recombination is poorly understood. Switch recombination occurs between pairs of switch regions located upstream of the constant heavy chain genes. Previously we showed that switch recombination breakpoints cluster to a defined subregion in the Sgamma3, Sgamma1 and Sgamma2b tandem repeats. We have developed a strategy for direct amplification of Smu/Sgamma3 composite fragments as well as Smu and Sgamma3 regions by PCR. This assay has been used to analyze the organization of Smu, Sgamma3 and a series of Smu/Sgamma3 recombination breakpoints from hybridomas and normal mitogen-activated splenic B cells. DNA sequence analysis of the switch fragments showed direct joining of Smu and Sgamma3 without deletions or duplications. Mutations were found in two switch junctions on both sides of the crossover point, suggesting that template switching is the most likely model for the mechanism of switch recombination. Statistical analysis of the positions of the recombination breakpoints in the Sgamma3 tandem repeat indicates the presence of two sub-clusters, suggesting non-random usage of DNA substrate in the recombination reaction. 相似文献
17.
18.
T H Mes P Kuperus J Kirschner J Stepanek P Oosterveld H Storchova J C den Nijs 《Génome》2000,43(4):634-641
Sequence variation in 2.2 kb of non-coding regions of the chloroplast genome of eight dandelions (Taraxacum: Lactuceae) from Asia and Europe is interpreted in the light of the phylogenetic signal of base substitutions vs. indels (insertions-deletions). The four non-coding regions displayed a total of approximately 30 structural mutations of which 9 are potentially phylogenetically informative. Insertions, deletions, and an inversion were found that involved consecutive stretches of up to 172 bases. When compared to phylogenetic relationships of the chloroplast genomes based on nucleotide substitutions only, many homoplasious indels (33%) were detected that differed considerably in length and did not comprise simple sequence repeats typically associated with replication slippage. Though many indels in the intergenic spacers were associated with direct repeats, frequently, the variable stretches participated in inverted repeat stabilized hairpins. In each intergenic spacer or intron examined, nucleotide stretches ranging from 30 to 60 bp were able to fold into stabilized secondary structures. When these indels were homoplasious, they always ranked among the most stabilized hairpins in the non-coding regions. The association of higher order structures that involve both classes of repeats and parallel structural mutations in hot spot regions of the chloroplast genome can be used to differentiate among mutations that differ in phylogenetic reliability. 相似文献
19.
Mutational analysis of the Tetrahymena telomerase RNA: identification of residues affecting telomerase activity in vitro. 总被引:4,自引:1,他引:3
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Telomere-specific repeat sequences are essential for chromosome end stability. Telomerase maintains telomere length by adding sequences de novo onto chromosome ends. The template domain of the telomerase RNA component dictates synthesis of species-specific telomeric repeats and other regions of the RNA have been suggested to be important for enzyme structure and/or catalysis. Using enzyme reconstituted in vitro with RNAs containing deletions or substitutions we identified nucleotides in the RNA component that are important for telomerase activity. Although many changes to conserved features in the RNA secondary structure did not abolish enzyme activity, levels of activity were often greatly reduced, suggesting that regions other than the template play a role in telomerase function. The template boundary was only altered by changes in stem II that affected the conserved region upstream of the template, not by changes in other regions, such as stems I, III and IV, consistent with a role of the conserved region in defining the 5' boundary of the template. Surprisingly, telomerase RNAs with substitutions or deletion of residues potentially abolishing the conserved pseudoknot structure had wild-type levels of telomerase activity. This suggests that this base pairing interaction may not be required for telomerase activity per se but may be conserved as a regulatory site for the enzyme in vivo. 相似文献
20.
Delagoutte E Goellner GM Guo J Baldacci G McMurray CT 《The Journal of biological chemistry》2008,283(19):13341-13356
Small insertions and deletions of trinucleotide repeats (TNRs) can occur by polymerase slippage and hairpin formation on either template or newly synthesized strands during replication. Although not predicted by a slippage model, deletions occur preferentially when 5'-CTG is in the lagging strand template and are highly favored over insertion events in rapidly replicating cells. The mechanism for the deletion bias and the orientation dependence of TNR instability is poorly understood. We report here that there is an orientation-dependent impediment to polymerase progression on 5'-CAG and 5'-CTG repeats that can be relieved by the binding of single-stranded DNA-binding protein. The block depends on the primary sequence of the TNR but does not correlate with the thermodynamic stability of hairpins. The orientation-dependent block of polymerase passage is the strongest when 5'-CAG is the template. We propose a "template-push" model in which the slow speed of DNA polymerase across the 5'-CAG leading strand template creates a threat to helicase-polymerase coupling. To prevent uncoupling, the TNR template is pushed out and by-passed. Hairpins do not cause the block, but appear to occur as a consequence of polymerase pass-over. 相似文献