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 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 Is the Principal Pathway of Homologous Recombination in Tandem Duplications of <Emphasis Type="Italic">Escherichia coli</Emphasis>

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.__________Translated from Genetika, Vol. 41, No. 8, 2005, pp. 1038–1044.Original Russian Text Copyright © 2005 by Prokop’ev, Sukhodolets.     

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 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.     

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.     

Effect of mutations for the ruvABC genes on recombination between direct DNA repairs in Escherichia coli strains carrying extended tandem duplication

The formation of haploid and diploid segregants was studied in Escherichia coli strains carrying heterozygous tandem duplications deoA deoB::Tn5/deoC deoD in the deoCABD operon region, in the genome of mutants for ruvABC genes. Homologous recombination in duplications of rec+ strains and in recBC sbcB, recQ and recF mutants, including those with blocks of both the RecBCD and RecF pathway, was shown in our previous work to be similar to adaptive mutagenesis: in this case, practically each cell forms a recombinant on a selective medium. In this work, mutants for ruv genes were found to differ in this respect, forming segregants at a frequency that was decreased by several orders of magnitude. These data confirm the conclusion that the genetic exchange in duplications proceeds through a special pathway of adaptive (or replicative) recombination connected with DNA replication. Upon selection of recombinants under conditions of thymine starvation, recombination cannot also be induced in ruv mutants. The recombinogenic effect of thymine starvation seems to occur at late stages of recombination, which are controlled by ruvABC genes.     

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.     

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.     

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.     

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.     

Effect of Mutations for the ruvABC Genes on Recombination between Direct DNA Repeats in Escherichia coli Strains Carrying Extended Tandem Duplications

The formation of haploid and diploid segregants was studied in Escherichia coli strains carrying heterozygous tandem duplications deoA deoB::Tn5/deoC deoD in the deoCABD operon region, in the genome of mutants forruvABCgenes. Homologous recombination in duplications of rec ⁺ strains and in recBC sbcB, recQand recF mutants, including those with blocks of both the RecBCD and RecF pathway, was shown in our previous work to be similar to adaptive mutagenesis: in this case, practically each cell forms a recombinant on a selective medium. In this work, mutants for ruv genes were found to differ in this respect, forming segregants at a frequency that was decreased by several orders of magnitude. These data confirm the conclusion that the genetic exchange in duplications proceeds through a special pathway of adaptive (or replicative) recombination connected with DNA replication. Upon selection of recombinants under conditions of thymine starvation, recombination cannot also be induced in ruv mutants. The recombinogenic effect of thymine starvation seems to occur at late stages of recombination, which are controlled by ruvABC 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.     

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.     

Principles of prokaryotic genome organization]

The peculiarities of bacterial chromosome organization are discussed, based mainly on the data on Escherichia coli. Highly important for bacterial genome organization is its division into two approx. equal half-genomes undergoing periodically "exchanges" of some kind displayed as continuous inversions including the oriC region of replication initiation. It is believed that short oligonucleotides are comprised in either of half-genomes. The former are predominantly oriented as direct repeats, which ensures the possibility of formation of tandem duplications consisting of identical genes--under conditions when selection for enhancing functions of corresponding genes takes place. Multiple tandem duplications capable of excision of plasmatic gene copies seem to initiate horizontal gene transfer in bacteria. Tandem gene duplications are probably being formed in the process of bacterial genetic recombination as well, when, as a result of non-equal crossing over, gene alleles derived from different strains are united into a tandem.     

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.     

Bacteriophage infection of minicells

Summary Off the transconjugants formed in theR. lupini conjugation 0.5 to 5% are merodiploids. When two differently pigmented parents are used in the crossing experiment the diploid transconjugants can be differentiated from the haploid recombinants by their additive pigmentation type. The segregation patterns of these diploid clones were analyzed. The results are in agreement with the theory that the exogenotic donor DNA can be integrated at different sites of the homologous recipient chromosomal region forming a tandem sequence. Consequently the segregants of these merodiploid clones are formed by endochromosomal recombination. This work was supported by the Deutsche Forschungsgemeinschaft and Stiftung Volkswagenwerk.     

Pathways for Homologous Recombination between Chromosomal Direct Repeats in Salmonella Typhimurium

Homologous recombination pathways probably evolved primarily to accomplish chromosomal repair and the formation and resolution of duplications by sister-chromosome exchanges. Various DNA lesions initiate these events. Classical recombination assays, involving bacterial sex, focus attention on double-strand ends of DNA. Sexual exchanges, initiated at these ends, depend on the RecBCD pathway. In the absence of RecBCD function, mutation of the sbcB and sbcC genes activates the apparently cryptic RecF pathway. To provide a more general view of recombination, we describe an assay in which endogenous DNA damage initiates recombination between chromosomal direct repeats. The repeats flank markers conferring lactose utilization (Lac(+)) and ampicillin resistance (Ap(R)); recombination generates Lac(-) Ap(S) segregants. In this assay, the RecF pathway is not cryptic; it plays a major role without sbcBC mutations. Others have proposed that single-strand gaps are the natural substrate for RecF-dependent recombination. Supporting this view, recombination stimulated by a double-strand break (DSB) in a chromosomal repeat depended on RecB function, not RecF function. Without RecBCD function, sbcBC mutations modified the RecF pathway and allowed it to catalyze DSB-stimulated recombination. Sexual recombination assays overestimate the importance of RecBCD and DSBs, and underestimate the importance of the RecF pathway.     

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.