Unequal crossing over is the principal pathway of homologous recombination in tandem duplications of Escherichia coli

Homologous recombination between direct DNA repeats in tandem duplications usually leads to their dissociation. An even number of crossovers between two copies of a duplication should lead to the formation of diploid segregants, i.e., to the preservation of the duplication. However, in studies of the genotype of diploid segregants in heterozygous tandem duplications of Escherichia coli, it was shown that they arise by unequal exchanges between sister chromosomes rather than by intrachromosomal exchanges. Generally, these exchanges lead to the establishment of the homozygous state of (heterozygous) duplications. Since the available data suggest that the exchange between sister chromosomes may be coupled with DNA replication, it is supposed that unequal exchanges between direct DNA repeats occur in the process of DNA replication.     

Unequal crossing-over in Escherichia coli

Unequal crossing-over between sister chromosomes in the process of DNA replication in Escherichia coli leads to the formation of tandem duplications, thus enhancing the activity of certain genes. In conjugational matings between genetically marked E. coli strains, unequal crossing-over leads to the formation of heterozygous tandem duplications. Studying these duplications as model systems allowed the conclusion that unequal crossing-over between direct DNA repeats of sister chromosomes is the main pathway of the formation of selected recombinants in E. coli strains carrying duplications. This was inferred from the data on the segregation of homozygous diploid recombinants by heterozygous duplications. Unequal crossing-over between sister chromosomes occurs as adaptive exchange providing the survival of the greater part of bacterial cells on a selective medium. The known phenomenon of adaptive mutagenesis may also be a consequence of unequal exchanges at the level of DNA mononucleotide repeats.     

Unequal crossing-over in Escherichia coli

Unequal crossing-over between sister chromosomes in the process of DNA replication in Escherichia coli leads to the formation of tandem duplications, thus enhancing the activity of certain genes. In conjugational matings between genetically marked E. coli strains, unequal crossing-over leads to the formation of heterozygous tandem duplications. Studying these duplications as model systems allowed the conclusion that unequal crossing-over between direct DNA repeats of sister chromosomes is the main pathway of the formation of selected recombinants in E. coli strains carrying duplications. This was inferred from the data on the segregation of homozygous diploid recombinants by heterozygous duplications. Unequal crossing-over between sister chromosomes occurs as adaptive exchange providing the survival of the greater part of bacterial cells on a selective medium. The known phenomenon of adaptive mutagenesis may also be a consequence of unequal exchanges at the level of DNA mononucleotide repeats.     

Unequal crossing over as the pathway of adaptive homologous recombination between the direct DNA repeats in tandem duplications of Escherichia coli

Homologous recombination between direct DNA repeats within the extended tandem duplications in E. coli results from unequal sister-chromosome exchanges. This conclusion follows from the observations on the segregation of completely or partly homozygous diploid segregants by heterozygous duplications. The formation of diploid segregants with preserved heterozygosity for the unselected markers could also result from "symmetrical" intrachromosomal recombination. Analysis of the segregant genotypes, however, confirmed their formation via unequal crossing over. The data obtained indicated that in tandem duplications segregation of diploid recombinants of different types was preceded by the formation of triplications as the products of unequal sister-chromosome exchanges. In heterozygous duplications, unequal crossing over is manifested as a highly frequent adaptive exchange, providing the survival of the most part of the duplication-carrying cells on selective medium. It is suggested that adaptive mutagenesis can be the consequence of unequal sister crossing over.     

Unequal Crossing Over as the Pathway of Adaptive Homologous Recombination between the Direct DNA Repeats in Tandem Duplications of Escherichia coli

Homologous recombination between direct DNA repeats within the extended tandem duplications in E. coli results from unequal sister-chromosome exchanges. This conclusion follows from the observations on the segregation of completely or partly homozygous diploid segregants by heterozygous duplications. The formation of diploid segregants with preserved heterozygosity for the unselected markers could also result from symmetrical intrachromosomal recombination. Analysis of the segregant genotypes, however, confirmed their formation via unequal crossing over. The data obtained indicated that in tandem duplications segregation of diploid recombinants of different types was preceded by the formation of triplications as the products of unequal sister-chromosome exchanges. In heterozygous duplications, unequal crossing over is manifested as a highly frequent adaptive exchange, providing the survival of the most part of the duplication-carrying cells on selective medium. It is suggested that adaptive mutagenesis can be the consequence of unequal sister crossing over.     

Unequal genetic exchange in Escherichia coli tandem duplications may represent a special pathway of homologous recombination

Heterozygous tandem duplications that appear in Escherichia coli conjugation matings segregate different types of haploid and diploid recombinants because of unequal crossing over between sister chromosomes. As shown previously, the frequency of segregants in the extended duplication D104 (approximately 150 kb or more than 3 min of the genetic map) heterozygous for E. coli deo-operon genes (deoA deoB::Tn5/deoC deoD) is not decreased in strains with defective RecBCD and RecF recombination pathways. Analysis of a shorter duplication of this type (approximately 46 kb) showed that the frequency of segregants in the strain recBC sbcBC recF was similar to that in a strain with undamaged system of recombination. Thus, genetic exchange between direct DNA repeats in tandem duplications may follow a special pathway of homologous recombination, which is independent of the recBC and recF genes.     

The importance of recombinations occuring in the process of DNA replication

In the process of DNA replication in Escherichia coli, the unequal crossing over occurs between DNA direct repeats in sister chromosomes. Such exchanges lead to the formation of tandem duplications and, therefore, to an increase of gene expression. It is proposed that non-homologous cohesion of sister chromosomes and unequal crossing over occur if the movement of replication fork is stopped. In the case of the unceasing movement of replication fork the homologous exchange between sister chromosomes occurs which provides their post-replication reparation.     

Role of recombination occurring during DNA replication

Unequal crossing over between direct DNA repeats of sister chromosomes occurs during DNA replication in Escherichia coli. Such exchanges yield tandem duplications and thereby increase the expression of the genes involved. Nonhomologous cohesion of sister chromosomes and unequal crossing over were assumed to take place when the replication fork stops. When the replication forks moves continuously, homologous exchanges between sister chromosomes ensure their postreplication repair.     

Unequal genetic exchange in <Emphasis Type="Italic">Escherichia coli</Emphasis> tandem duplications may represent a special pathway of homologous recombination

Heterozygous tandem duplications that appear in Escherichia coli conjugation matings segregate different types of haploid and diploid recombinants formed by unequal crossing over between sister chromosomes. As shown previously, the frequency of segregants in the extended duplication D104 (150 kb or more than 3 min of the genetic map) heterozygous for E. coli deo-operon genes (deoA deoB::Tn5/deoC deoD) is not decreased in strains with defective RecBCD and RecF recombination pathways. Analysis of a shorter duplication of this type (46 kb) showed that the frequency of segregants in the strain recBC sbcBC recF was similar to that in a strain with undamaged system of recombination. Thus, genetic exchange between direct DNA repeats in tandem duplications may follow a special pathway of homologous recombination, which is independent of the recBC and recF genes.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 307–311.Original Russian Text Copyright © 2005 by Sukhodolets, Prokopev.     

Homologous recombination and chromosomal rearrangements in Escherichia coli strains carrying a heterozygous tandem duplication]

Heterozygous tandem duplications formed in conjugational matings in Escherichia coli provides a convenient model system for studying the evolution of bacterial chromosome. Heterozygous duplications segregate various classes of haploid and diploid recombinants that appear as a result of unequal crossing over between sister chromosomes. In this work, an extended tandem duplication in the deo operon of E. coli carrying deoA deoB::Tn5/deoC deoD thr::Tn9 alleles was examined. Recombination between homologous DNA repeats in the duplication was studied in strains carrying different combinations of recBC, sbcBC, recB::Tn10, recQ::Tn3 mutations. The frequency of recombination between homologous DNA repeats was very high in all strains and did not decrease when the RecBCD and RecF recombinational pathways were simultaneously damaged in strains with the recB sbcBC recQ (or recF) genotype. It is assumed that unequal crossing over between direct DNA repeats in duplications may proceed through a particular pathway of "adaptive" recombination.     

Homologous recombination between direct repeats of chromosomal segments comprising heterozygotic duplications in Escherichia coli

In conjugational matings between double mutants for the deo operon of Escherichia coli, haploid recombinants and extended tandem duplications deoC deoD/deoA deoB::Tn5 with the DeoC+DeoA+DeoB+DeoD- phenotype are formed (the deoD+ allele is not expressed due to the polar effect of the Tn5 insertion). Selection for the expression of the recessive deoC deoD alleles (in the thyA genome) leads to the segregation of haploid clones by duplications and also of clones that retain the diploidy but that are homozygous for deoC deoD. In addition to haploids, diploid clones retaining the duplications have also been found among the DeoD+ segregants. The phenotype of segregants retaining the duplication shows that they were formed by an unequal exchange between sister chromosomes. A comparison of segregation frequency of haploid and diploid DeoD+ clones in rec+ and recBC sbcB sbcC strains shows that duplications in the rec+ genome are more stable. On this basis, it is assumed that the RecBCD pathway possibly makes a greater contribution than the RecF pathway to the preservation of heterozygous duplications playing an important role in the evolution of prokaryotes.     

The function of recombinations occurring in the process of DNA replication in Escherichia coli

In a number of works dealing with the relationship between replication and recombination in bacteria, it is assumed that recombinations permit the replication forks to resume moving after having stopped at the damage sites of the template DNA. As an evidence for recombination occurring during DNA replication, the involvement in this process of proteins RuvABC and RecG, providing processing of the Holliday junctions after recombination, is considered. However, it has been shown that these proteins are not essential for resuming DNA synthesis after an exposure of bacteria to UV light. These data cast doubt on the necessity of recombination for reactivation of replication initiated in the oriC region. Studying recombination in tandem duplications in Escherichia coli showed that during replication, unequal crossing over occurs between direct DNA repeats of sister chromosomes. In wild strains, this crossing over results in tandem duplications, thereby enhancing the expression of certain genes. Thus, recombination of two types occurs during DNA replication: unequal crossing over leading to duplications and homologous exchange, responsible for post-replication DNA repair. The unequal exchange constitutes a component of SOS response of the cell to deterioration of the environment.     

Recombinations occurring in the process of DNA replication in Escherichia coli

In a number of works dealing with the relationship between replication and recombination in bacteria, it is assumed that recombinations permit the replication forks to resume moving after having stopped at the damage sites of the template DNA. As an evidence for recombination occurring during DNA replication, the involvement in this process of proteins RuvABC and RecG, providing processing of the Holliday junctions after recombination, is considered. However, it has been shown that these proteins are not essential for resuming DNA synthesis after an exposure of bacteria to UV light. These data cast doubt on the necessity of recombination for reactivation of replication initiated in the oriC region. Studying recombination in tandem duplications in Escherichia coli showed that during replication, unequal crossing over occurs between direct DNA repeats of sister chromosomes. In wild strains, this crossing over results in tandem duplications, thereby enhancing the expression of certain genes. Thus, recombination of two types occurs during DNA replication: unequal crossing over leading to duplications and homologous exchange, responsible for post-replication DNA repair. The unequal exchange constitutes a component of SOS response of the cell to deterioration of the environment.     

Study of Homologous Recombination and Chromosomal Rearrangements inEscherichia coliStrains Carrying a Heterozygous Tandem Duplication

Heterozygous tandem duplications formed in conjugational matings in Escherichia coliprovides a convenient model system for studying the evolution of bacterial chromosome. Heterozygous duplications segregate various classes of haploid and diploid recombinants that appear as a result of unequal crossing over between sister chromosomes. In this work, an extended tandem duplication in the deooperon of E. colicarrying deoA deoB::Tn5/deoC deoD thr::Tn9alleles was examined. Recombination between homologous DNA repeats in the duplication was studied in strains carrying different combinations of recBC, sbcBC, recB::Tn10, recQ::Tn3and recF::Tn3mutations. The frequency of recombination between homologous DNA repeats was very high in all strains and did not decrease when the RecBCD and RecF recombinational pathways were simultaneously damaged in strains with the recB sbcBC recQ(or recF) genotype. It is assumed that unequal crossing over between direct DNA repeats in duplications may proceed through a particular pathway of adaptive recombination.     

Studying recombination in heterozygous tandem duplications in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Our previous data showed that the principal pathway of the formation of selected recombinants in Escherichia coli strains carrying heterozygous tandem duplications is unequal crossing over between sister chromosomes. Data presented in this work showed that when DNA homology is not disturbed (due to transposon insertion), intragenic recombinants can occur directly in the region of recombination through intrachromomal exchange as well.     

Studying recombination in heterozygous tandem duplications in Escherichia coli

Our previous data showed that the principal pathway of the formation of selected recombinants in Escherichia coli strains carrying heterozygous tandem duplications is unequal crossing over between sister chromosomes. Data presented in this work showed that when DNA homology is not disturbed (due to transposon insertion), intragenic recombinants can occur directly in the region of recombination through intrachromomal exchange as well.     

Multiple Pathways of Duplication Formation with and Without Recombination (RecA) in Salmonella enterica

Duplications are often attributed to “unequal recombination” between separated, directly repeated sequence elements (>100 bp), events that leave a recombinant element at the duplication junction. However, in the bacterial chromosome, duplications form at high rates (10⁻³–10⁻⁵/cell/division) even without recombination (RecA). Here we describe 1800 spontaneous lac duplications trapped nonselectively on the low-copy F′₁₂₈ plasmid, where lac is flanked by direct repeats of the transposable element IS3 (1258 bp) and by numerous quasipalindromic REP elements (30 bp). Duplications form at a high rate (10⁻⁴/cell/division) that is reduced only about 11-fold in the absence of RecA. With and without RecA, most duplications arise by recombination between IS3 elements (97%). Formation of these duplications is stimulated by IS3 transposase (Tnp) and plasmid transfer functions (TraI). Three duplication pathways are proposed. First, plasmid dimers form at a high rate stimulated by RecA and are then modified by deletions between IS3 elements (resolution) that leave a monomeric plasmid with an IS3-flanked lac duplication. Second, without RecA, duplications occur by single-strand annealing of DNA ends generated in different sister chromosomes after transposase nicks DNA near participating IS3 elements. The absence of RecA may stimulate annealing by allowing chromosome breaks to persist. Third, a minority of lac duplications (3%) have short (0–36 bp) junction sequences (SJ), some of which are located within REP elements. These duplication types form without RecA, Tnp, or Tra by a pathway in which the palindromic junctions of a tandem inversion duplication (TID) may stimulate deletions that leave the final duplication.     

Mechanisms of tandem duplication in the Duchenne muscular dystrophy gene include both homologous and nonhomologous intrachromosomal recombination.

Three tandem duplications were previously identified in patients with Duchenne muscular dystrophy and were shown in each case to have a subset of dystrophin gene exons duplicated. The origin of these duplications was traced to the single X chromosome of the maternal grandfathers, suggesting that an intrachromosomal event (unequal sister chromatid exchange) was involved in the formation of these duplications. In the present study, a DNA segment containing the duplication junction and the normal DNA that corresponds to both ends of the duplicated region have been cloned. Subsequent mapping studies confirmed the tandem arrangement (head to tail) of these duplications and revealed their sizes to be 130 kb, approximately 300 kb, and 35-80 kb, respectively. Sequence analysis of the duplication junctions showed that one duplication was due to homologous recombination between two repetitive elements (Alu sequences) and the other two were due to recombination between unrelated nonhomologous sequences. In the latter cases, the preferred cleavage sites of the eukaryotic type I and II DNA topoisomerases were found at the junctions of these duplications, suggesting a possible role of these enzymes in the chromatid exchange events. This study provides the first insight into the molecular basis of gene duplications formed through unequal sister chromatid exchange in humans.     

Techniques for Manipulating Chromosomal Rearrangements and Their Application to DROSOPHILA MELANOGASTER. I. Pericentric Inversions

Techniques have been developed for manipulating pericentric inversions in Drosophila that are based on the lethality of grossly aneuploid zygotes and the existence of recombinationally interconvertible genotypes for any heterozygous inversion complex: males of some of these genotypes will produce only aneuploid sperm, which can be used to rescue complementary aneuploid ova and selectively recover recombinational derivatives of inversions. Markers can be recombined into inversions through a sequence of selected single exchanges, and a novel type of duplication can be synthesized from overlapping inversions that has the characteristics of both insertional and tandem duplications; there are also applications to half-tetrad analyses.——Two cytogenetic screens are developed: (1) the dominant lethality of a large insertional-tandem duplication can be reverted by deletional events that give rise to net deficiencies or duplications, and (2) deficiencies and tandem duplications in proximal regions can be selectively recovered as the results of unequal exchanges within an inversion loop. Recombinants have been recovered between breakpoints separated by distances of as little as fifty bands, arguing against the existence of some small number of sites necessary for the initiation of recombinational pairing. In several instances, hyperploids for four to six numbered divisions were observed to be fertile in both sexes.