Sister Strand Exchange Research Articles

Genetic instabilities of (CCTG)·(CAGG) and (ATTCT)·(AGAAT) disease‐associated repeats reveal multiple pathways for repeat deletion

The DNA repeats (CTG).(CAG), (CGG).(CCG), (GAA).(TTC), (ATTCT).(AGAAT), and (CCTG).(CAGG), undergo expansion in humans leading to neurodegenerative disease. A genetic assay for repeat instability has revealed that the activities of RecA and RecB during replication restart are involved in a high rate of deletion of (CTG).(CAG) repeats in E. coli. This assay has been applied to (CCTG).(CAGG) repeats associated with myotonic dystrophy type 2 (DM2) that expand to 11 000 copies and to spinocerebellar ataxia type 10 (SCA10) (ATTCT).(AGAAT) repeats that expand to 4500 copies in affected individuals. DM2 (CCTG).(CAGG) repeats show a moderate rate of instability, less than that observed for the myotonic dystrophy type 1 (CTG).(CAG) repeats, while the SCA10 (ATTCT).(AGAAT) repeats were remarkably stable in E. coli. In contrast to (CTG).(CAG) repeats, deletions of the DM2 and SCA10 repeats were not dependent on RecA and RecB, suggesting that replication restart may not be a predominant mechanism by which these repeats undergo deletion. These results suggest that different molecular mechanisms, or pathways, are responsible for the instability of different disease-associated DNA repeats in E. coli. These pathways involve simple replication slippage and various sister strand exchange events leading to deletions or expansions, often associated with plasmid dimerization. The differences in the mechanisms of repeat deletion may result from the differential propensity of these repeats to form various DNA secondary structures and their differential proclivity for primer-template misalignment during replication.

A mechanism for gene conversion in fungi

A mechanism for gene conversion is proposed which overcomes many of the difficulties that any copy choice model encounters. It is suggested that along with general genetic pairing of homologous genomes at meiosis, effective pairing over short regions of the genetic material occurs at the molecular level by the separation of the strands of the DNA double helices, followed by the annealing of strands from two homologous chromatids. If the annealed region happens to span a heterozygous site, mispairing of bases will occur. Such a situation may be analogous to that in DNA which is damaged by mutagens; the same or similar repair mechanisms may operate, and these, by adjusting the base sequences in order to restore normal base pairing, would bring about gene conversion in the absence of any genetic replication. The model indicates how precise breakage and rejoining of chromatids could occur in the vicinity of the conversion, so that conversion would frequently be accompanied by the recombination of outside markers. The model also proposes that the distance between two mutant sites on a fine structure map depends not so much on the frequency of a recombinational event occurring between them, but rather on the degree of inhibition of the processes of genetic pairing by the mutants themselves. The model will explain almost all the data in a formal way, and it has the advantage over copy choice mechanisms for gene conversion in (1) being compatible with semi-conservative replication of DNA, (2) not invoking DNA synthesis during or after genetic pairing, (3) providing a molecular mechanism for close specific pairing, (4) making it unnecessary to postulate sister strand exchange or a process akin to this, (5) suggesting why rates of gene conversion in opposite directions are sometimes unequal and (6) providing an explanation of the clustering of mutant sites, a basis for map expansion and for the apparently capricious departure of fine structure maps from additivity. Although the model proposed is a general rather than a specific one, it suggests that the process of conversion and intragenic recombination is more complex than is usually believed, since it depends on several interacting factors. Nevertheless, it is hoped that the introduction of a model with this complexity will help to stimulate specific experiments, and that these will provide definitive information which would never be obtained if simpler models of conversion and intragenic recombination were believed to explain the genetic data sufficiently well.

Slipped misalignment mechanisms of deletion formation: analysis of deletion endpoints

To gain insight into the mechanisms of deletion formation between tandem repeats, Escherichia coli plasmids were engineered to carry a 101 bp tandem duplication within the tetA gene such that deletion of one of the repeats restores an intact tetA gene and tetracycline resistance to the cell. Four base-pair changes were introduced into one of the tandem repeats to serve as genetic markers. After selection for deletion, individual plasmid products were sequenced to deduce where within the repeat the deletion had occurred. Our analysis shows most deletions are fusions of the two repeats in a single 20 bp interval. This is consistent with the simple replication slip-pair model for deletion formation and suggests that this interval may have unusual features that promote deletion. Dimer replicon products have experienced a sister-chromosome exchange event in addition to deletion and carry two tetA loci: a deleted locus showing a similar distribution of endpoints as seen in the monomer products and an unchanged repeat locus. Seemingly reciprocal dimers are occasionally recovered which carry both a deleted and a triplicated tetA locus. These are not truly reciprocal in that the sequence analysis showed that the deletion and triplication had occurred in separate intervals. Sequence analysis of the dimeric products is consistent with predictions from our sister-strand exchange model where slipped alignment of nascent DNA strands induces deletion formation concomitant with sister-chromosome exchange.

Enhanced deletion formation by aberrant DNA replication in Escherichia coli.

Repeated genes and sequences are prone to genetic rearrangements including deletions. We have investigated deletion formation in Escherichia coli strains mutant for various replication functions. Deletion was selected between 787 base pair tandem repeats carried either on a ColE1-derived plasmid or on the E. coli chromosome. Only mutations in functions associated with DNA Polymerase III elevated deletion rates in our assays. Especially large increases were observed in strains mutant in dnaQ the epsilon editing subunit of Pol III, and dnaB, the replication fork helicase. Mutations in several other functions also altered deletion formation: the alpha polymerase (dnal;), the gamma clamp loader complex (holC, dnaX), and the beta clamp (dnaN) subunits of Pol III and the primosomal proteins, dnaC and priA. Aberrant replication stimulated deletions through several pathways. Whereas the elevation in dnaB strains was mostly recA- and lexA-dependent, that in dnaQ strains was mostly recA- and lexA-independent. Deletion product analysis suggested that slipped mispairing, producing monomeric replicon products, may be preferentially increased in a dnaQ mutant and sister-strand exchange, producing dimeric replicon products, may be elevated in dnaE mutants. We conclude that aberrant Polymerase III replication can stimulate deletion events through several mechanisms of deletion and via both recA-dependent and independent pathways.

Recombination between chromosomal IS200 elements supports frequent duplication formation in Salmonella typhimurium.

Spontaneous tandem chromosomal duplications are common in populations of Escherichia coli and Salmonella typhimurium. They range in frequency for a given locus from 10(-2) to 10(-4) and probably form by RecA-dependent unequal sister strand exchanges between repetitive sequences in direct order. Certain duplications have been observed previously to confer a growth advantage under specific selective conditions. Tandem chromosomal duplications are unstable and are lost at high frequencies, representing a readily reversible source of genomic variation. Six copies of a small mobile genetic element IS200 are evenly distributed around the chromosome of S. typhimurium strain LT2. A survey of 120 independent chromosomal duplications (20 for each of six loci) revealed that recombination between IS200 elements accounted for the majority of the duplications isolated for three of the loci tested. Duplications of the his operon were almost exclusively due to recombination between repeated IS200 elements. These data add further support to the idea that mobile genetic elements provide sequence repeats that play an important role in recombinational chromosome rearrangements, which may contribute to adaptation of bacteria to stressful conditions.

A sister-strand exchange mechanism for recA-independent deletion of repeated DNA sequences in Escherichia coli.

In the genomes of many organisms, deletions arise between tandemly repeated DNA sequences of lengths ranging from several kilobases to only a few nucleotides. Using a plasmid-based assay for deletion of a 787-bp tandem repeat, we have found that a recA-independent mechanism contributes substantially to the deletion process of even this large region of homology. No Escherichia coli recombination gene tested, including recA, had greater than a fivefold effect on deletion rates. The recA-independence of deletion formation is also observed with constructions present on the chromosome. RecA promotes synapsis and transfer of homologous DNA strands in vitro and is indispensable for intermolecular recombination events in vivo measured after conjugation. Because deletion formation in E. coli shows little or no dependence on recA, it has been assumed that homologous recombination contributes little to the deletion process. However, we have found recA-independent deletion products suggestive of reciprocal crossovers when branch migration in the cell is inhibited by a ruvA mutation. We propose a model for recA-independent crossovers between replicating sister strands, which can also explain deletion or amplification of repeated sequences. We suggest that this process may be initiated as post-replicational DNA repair; subsequent strand misalignment at repeated sequences leads to genetic rearrangements.

Recombination between homologies in direct and inverse orientation in the chromosome of Salmonella: intervals which are nonpermissive for inversion formation.

Sequences placed in inverse order at particular chromosome sites (permissive) recombine to generate an inversion; the same sequences, placed at other sites (nonpermissive) interact recombinationally but do not form the expected inversion recombinants. We have investigated the events that occur between sequences at nonpermissive sites. Genetically marked lac operons in inverse order were placed at nonpermissive sites in a single chromosome and Lac+ recombinants were selected. No inversions were formed. The Lac+ recombinants recovered include double-recombinant types in which information appears to have undergone a nonreciprocal information exchange; one mutant copy is repaired with no alteration of the other copy. Recombination within the lac operon is stimulated more than 100-fold by the presence of extensive homology (antenna sequences) outside of the region for which recombination is selected. Sequences placed in direct order at the ends of the same noninvertible chromosome segment recombine to form all the expected recombinant types including those in which a reciprocal exchange has generated a duplication. All the detected recombinant types can be accounted for by recombination between sister chromosomes. These results are discussed in terms of two alternative models. One explanation of the failure to detect inversion of some intervals is that particular inversions are lethal, despite the fact that no essential sequences are disrupted. Another explanation is that chromosome topology prevents sequences at nonpermissive sites in a single chromosome from engaging in the direct interaction required for inversion formation, but allows the sister strand exchanges that can generate the recombinant observed.

One-step and stepwise magnification of a bobbed lethal chromosome in Drosophila melanogaster.

Bobbed lethal (bbl) chromosomes carry too few ribosomal genes for homozygous flies to be viable. Reversion of bbl chromosomes to bb or nearly bb+ occurs under magnifying conditions at a low frequency in a single generation. These reversions occur too rapidly to be accounted for by single unequal sister chromatid exchanges and seem unlikely to be due to multiple sister strand exchanges within a given cell lineage. Analysis of several one-step revertants indicates that they are X-Y recombinant chromosomes which probably arise from X-Y recombination at bb. The addition of ribosomal genes from the Y chromosome to the bbl chromosome explains the more rapid reversion of the bbl chromosome than is permitted by single events of unequal sister chromatid exchange. Analysis of stepwise bbl magnified chromosomes, which were selected over a period of 4-9 magnifying generations, shows ribosomal gene patterns that are closely similar to each other. Similarity in rDNA pattern among stepwise magnified products of the same parental chromosome is consistent with reversion by a mechanism of unequal sister strand exchange.

Ring chromosomes and rDNA magnification in Drosophila.

Tartof showed that ribosomal gene magnification in Drosophila was inhibited in a ring X chromosome. The present studies extend this observation by showing that ring X chromosomes are lost meiotically in male Drosophila undergoing ribosomal gene magnification as evidenced by the recovery of a lower number of ring-bearing progeny under magnifying conditions compared with nonmagnifying conditions. Associated with ring chromosome loss is a highly significant increase in the number of double-sized dicentric ring chromosomes in meiotic cells from magnifying males. These observations explain the failure of ring X chromosomes to magnify and imply that magnification in rod chromosomes occurs via a mechanism of unequal sister chromatid exchange. Our results support the hypothesis that the primary event of magnification is a sister chromatid exchange in the rDNA, that the frequency of sister strand exchanges is increased in magnifying flies, that a significant number of exchanges in magnifying flies occurs meiotically and that some of the exchanges are nonreciprocal. We have also found that autosomal mutations can affect both the frequency of abnormal ring structures and the ability of ring X chromosomes to magnify.

Genetic properties of chromosomally integrated 2 mu plasmid DNA in yeast.

We obtained strains of yeast with large segments of 2 mu plasmid DNA integrated at several chromosomal locations by selecting genetically for recombination between a chromosomal sequence carried on a 2 mu-circle-containing hybrid plasmid and a homologous sequence on the chromosome. In all diploids examined, the presence of 2 mu circle sequences causes a marked instability of the chromosome into which the 2 mu DNA is inserted. Although in some cases the loss of genetic markers is due to physical loss of the entire chromosome, in most cases the loss of markers appears to be due to a mitotic homozygotization of markers: the allelic information from the homologous chromosome replaces the information distal to the integrated 2 mu DNA. The instability caused by integrated 2 mu DNA sequences requires the activity of the specialized site-specific recombination system encoded by the 2 mu plasmid. We propose that the presence of integrated 2 mu DNA allows efficient integration of additional copies of the intact 2 mu plasmid by the action of the plasmid-coded special recombination system. Unequal sister-strand exchanges within the inverted repetition would result in the formation of dicentric chromosomes whose breakage during mitosis might begin a cycle analogous to the breakage-fusion-bridge cycle described many years ago in maize.

Mechanism of E. coli RecA protein directed strand exchanges in post-replication repair of DNA.

Escherichia coli mutants carrying recA− are both recombination deficient and unable to perform post-replication repair1,2. The product of the recA gene regulates the inducible DNA repair functions (the SOS response to DNA damage)3,4, and is directly involved in homologous pairing5–8 and strand exchange9–12, two reactions fundamental to recombination and post-replication repair. The filling of post-replication gaps is thought to occur by homologous pairing of the gapped DNA duplex with an intact duplex, followed by cutting of the intact molecule so that sister strand exchanges can take place. Using in vitro systems, we have shown previously that purified RecA protein binds cooperatively to duplex DNA that contains gaps13, and promotes joint molecule formation (synapsis) between gapped and intact duplexes7,8. Moreover, RecA protein promotes a reciprocal exchange of strands between paired DNA molecules10,12,14. Here, we investigate the mechanism of sister strand exchange thought to occur during post-replication repair. We show that RecA protein initiates strand exchange from a nicked duplex, transferring the 3′-OH terminus at the nick into the single-stranded (ss) region of the gapped molecule. In the presence of ATP, two heteroduplex molecules are formed as RecA protein drives the reciprocal exchanges in one direction starting at the site of the original crossover.

Induction of pure and sectored mutant clones in excision-proficient and deficient strains of yeast

We have found that UV-induced mutation frequency in a forward non-selective assay system (scoring white adex ade2 double auxotroph mutants among the red pigmented ade2 clones) increases linearly with dose up to a maximum frequency of about 3 × 10 −3 mutants per survivor and then declines in both RAD wild-type and rad2 excision deficient strains of Saccharomyces cerevisiae. Mutation frequencies of the RAD and the rad2 strains plotted against survival are nearly identical over the entire survival range. On this basis we conclude that unexcised pyrimidine dimers are the predominant type of pre-mutational lesions in both strains. In the RAD wild-type strain pure mutant clones outnumber sectors in a 10 : 1 ratio at all doses used; in rad2 this ratio varies from 1 : 1 at low doses up to 10 : 1 at high doses. As others have concluded for wild-type strains we find also in the rad2 strain that pure clone formation cannot be accounted for quantitatively by lethal sectoring events alone. We conclude that heteroduplex repair is a crucial step in pure mutant clone formation and we examine the plausibility of certain macromolecular mechanisms according to which heteroduplex repair may be coupled with replication, repair and sister strand exchange in yeast mutagenesis.

One way to exclude sister strand exchange in meiotic recombination.

DOES sister strand exchange occur as a normal feature during meiosis, or are meiotic crossovers restricted to non-sister chromatids? Evidence from experimental cytology shows that the contribution of sister strand exchange to meiotic recombination is insignificant1,2, or well below the values expected from chiasma frequencies3–6, indicating that sister strand exchange is generally discriminated against in meiotic recombination. Considering fragmentary knowledge from various experimental sources, I suggest how such discrimination may occur.

The effect of colchicine on subchromatid exchanges in root meristem cells of Vicia faba and Allium cepa

The problem of the duplication of subchromatid exchanges has been studied in Vicia faba and Allium cepa root meristems, respectively, with 8-ethoxycaffeine (EOC) and 6-methylcoumarin (6-MC), both effective in the DNA postsynthetic phases (G 2 and early prophase). A high frequency of subchromatid exchanges was induced by the mutagens at 2 h recovery after treatment; both species showed chromatid exchanges at the T 2 divisions and chromosome aberrations at the T 3 divisions after treatments. On the contrary, when colchicine was given to EOC-or 6-MC-treated roots in order to label the cells containing subchromatid exchanges, only chromosome-type aberrations were observed in both species at the T 2 (tetraploid) division. Assuming that sister-strand exchanges are crossover-like spontaneous events, an hypothesis is formulated to explain the presence of chromosome aberrations in T 2 (tetraploid) cells.

Evidence for nonrandom rejoining of chromatid breaks and its relation to the origin of sister-chromatid exchanges

Studies were made of the relative frequencies of sister-strand exchanges, isochromatid deletions, and symmetrical and asymmetrical interchanges involving daughter endoreduplicated chromosomes (diplochromosomes) in tritiated thymidine-labeled aneuploid Chinese hamster and diploid human cells. It was found that the reunion of broken ends in the endoreduplicated chromosomes was not random and that the ratio of symmetrical to asymmetrical interchanges was about the same as the ratio of sister-strand exchanges to isochromatid deletions. These data are interpreted to mean that sister-strand exchanges may be radiation-induced exchanges resulting from isolocus breaks that do not rejoin in the expected random fashion. The very high frequency of interchanges in daughter endoreduplicated chromosomes is taken as evidence in support of the site hypothesis of Wolff and Atwood (see ref. 19).

A more critical test of hypotheses of crossing over which involve sister-strand exchange.

A more critical test of hypotheses of crossing over which involve sister-strand exchange.

Are sister chromatid exchanges sister strand crossovers or radiation-induced exchanges?

Abstract From experiments that gave the ratio of twin sister strand exchanges to single sister strand exchanges in labeled chromosomes of the second division following labeling, Taylor had found that the individual units of chromatids exhibited polarity. It was thought that the events that led to these exchanges were sister strand crossovers. The ratios of twins to single exchanges, however, give far better fits to the yields expected if these exchanges were in reality radiation induced. Radiation-induced chromatid exchanges can also account for the type of isolabeling observed in the experiments of Peacock without the invocation of a multistranded chromosome.

Compound loci and coincident mutation in Neurospora.

To explain the high frequency of partial and intermediate back-mutants and the back-mutation of deletions, a model of mutation production based on unequal sister strand exchange in multi-repeat loci is proposed.

THE ANALYSIS OF ROD DERIVATIVES OF AN UNSTABLE RING CHROMOSOME OF DROSOPHILA MELANOGASTER

In contrast to those of corn, the ring chromosomes of Drosophila melanogaster are, with one exception, relatively stable. This exceptional Drosophila ring, called wVC, is derived from a stable ring-X chromosome, and its instability is manifested through the frequent production of gynandromorphs, XO males, and dominant lethals among prospective wuc/rod progeny. It has been proposed that these manifestations of wvC instability are a consequence of anaphase bridge formation by the wvc chromosome, whereby gynandromorphs and XO males arise from bridge loss, and dominant lethals owe their origin to bridge breakage (HINTON 1955). Since the work of RICCLINTOCK (1938) and SCHWARTZ (1953) suggests that sister-strand exchange is responsible for anaphase bridge formation by the ring chromosomes of corn, it appears that the same process might be the cause of wVc instability. If this is true, the manifestations of instability should disappear upon conversion of the unstable wvc ring chromosome to a rod chromosome because ring structure is a prerequisite for anaphase bridge formation as a consequence of sister-strand exchange. Studies on rod-wUC chromosomes derived from unstable ring-wvc chromosomes, as will be described in this paper, show that these rods are unstable. Although the manifestations of instability by wuc rods are qualitatively different from those of the unstable ring-wvc chromosome, they nevertheless are capable of interpretation in terms of anaphase bridge formation.

DOES SISTER-STRAND CROSSING OVER OCCUR IN DROSOPHILA MELANOGASTER

The problem of whether sister-strand crossing over does or does not occur is of some importance for general theories of crossing over. In most genetic experiments, i t is not possible to decide whether sister-strand exchange has or has not occurred because the results of both alternatives are indistinguishable. But with the use of the closed-X chromosome, described by L. V. MORGAN (1933), a solution of this question seems possible. In heterozygous condition, the closed-X chromosome gives no off spring coming from single or triple crossing over, and there is a corresponding and fairly large proportion of eggs that fail to hatch. When total crossing over is reduced by the use of an inversion-bearing chromosome opposite the closed-X, there is observed a reduction in the frequency of inviable eggs. It was inferred (loc. cit.) that in meiosis, single and triple exchanges between a closed-X and a normal X result in chromosome attachments which give these inviable eggs. Hence, in the presence of an inversion which reduces the frequency of crossing over, fewer single and triple crossings over occur to give inviable eggs. In this case, especially if the suppression of crossing over is almost complete, the chief causes of inviable eggs would be (a) the standard inviability of the stocks, and (b) sister-strand crossing over between closed-X chromosomes. With adequate precautions and controls, the standard residual inviability could be estimated, and a direct measure obtained of the frequency of sister-strand crossing over. The writers wish to acknowledge the kindness of L. V. MORGAN in making available stocks of the closed-X line for this experiment. One of us (N.K.) is also indebted to the DYCKMAN FUND of COLUMBIA UNIVERSITY for a grant during the summer of 1934.