Excision Of Tn10 Research Articles

Role of DNA Polymerases in Repeat-Mediated Genome Instability

Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreich's ataxia (GAA)(n) repeats in a yeast experimental system. The elementary step of expansion corresponded to ~160 bp in the wild-type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase α was mutated, suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM). Notably, defects in the replicative DNA polymerases δ and ε strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase δ mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism.

Genetic characterization of Escherichia coli RecN protein as a member of SMC family of proteins

The proteins of SMC family are characterized by having Walker A and B sites. The Escherichia coli RecN protein is a prokaryotic member of SMC family that involved in the induced excision of Tn10 and the repair of the DNA double strand breaks. In this work, the Walker A nucleotide binding site of the E. coli RecN protein was mutated by changing the highly conserved lysine residue 35 to the aspartic acid (D), designated as recNK35D. Reverse genetics was utilized to delete the entire recN gene (ΔrecN108) or introduce the recNK35D gene into the E. coli chromosomal DNA. The recNK35D cells showed decrease in the frequency of excision of Tn10 from gal76::Tn10 after treatment with mitomycin C compared to recN+ cells. The ΔrecN108 cells showed an un-induced increase in the frequency of Tn10 excision from gal76::Tn10 in rec+ background while, recBC sbcBC ΔrecN108 cells are completely deficient in Tn10 excision. The recombination proficiency is reduced in cells carrying recBC sbcBC cells in addition recNK35D mutation. We observed that the Walker A nucleotide binding site is important for the RecN protein. Strains that deleted recN gene are recombination deficient and more sensitive to mitomycin C than strains carrying recNK35D.

Precise excision of Tn5 in Escherichia coli K12 mutants with alterations in common component of phosphotransferase system and in DNA-gyrase subunits

The effect of ptsH, gyrA and gyrB mutations on the frequency of precise excision (PE) of transposon Tn5 was studied in Escherichia coli K12. The conjugative plasmid with Tn5 integrated in the tet gene of Tn10 was used as a model in the experiments. It was shown that mutational damage to the HPr protein (ptsH mutation), a common component of the bacterial PEP-dependent phosphotransferase system (PTS), or changes in the DNA-gyrase A subunit (gyrA mutation) increased the frequency of PE. The gyrB mutation (mutational damage of DNA-gyrase B subunit) had almost no influence on PE frequency. The presence of glucose or fructose in growth media resulted in enhanced PE frequency, while the insertion of ptsH or gyrA mutations under the same conditions caused a decrease in the PE frequency. gyrB mutants had no similar effects. The demonstrated data indicate the need for the intact PTS for the balanced supercoiled DNA state maintained by DNA gyrase.

ATP Hydrolysis Is Essential for the Function of the Uup ATP-binding Cassette ATPase in Precise Excision of Transposons

In Escherichia coli K-12, the RecA- and transposase-independent precise excision of transposons is thought to be mediated by the slippage of the DNA polymerase between the two short direct repeats that flank the transposon. Inactivation of the uup gene, encoding an ATP-binding cassette (ABC) ATPase, led to an important increase in the frequency of precise excision of transposons Tn10 and Tn5 and a defective growth of bacteriophage Mu. To provide insight into the mechanism of Uup in transposon excision, we purified this protein, and we demonstrated that it is a cytosolic ABC protein. Purified recombinant Uup binds and hydrolyzes ATP and undergoes a large conformational change in the presence of this nucleotide. This change affects a carboxyl-terminal domain of the protein that displays predicted structural homology with the socalled little finger domain of Y family DNA polymerases. In these enzymes, this domain is involved in DNA binding and in the processivity of replication. We show that Uup binds to DNA and that this binding is in part dependent on its carboxyl-terminal domain. Analysis of Walker motif B mutants suggests that ATP hydrolysis at the two ABC domains is strictly coordinated and is essential for the function of Uup in vivo.

Stationary phase deletions in Escherichia coli: II. Mutations which stimulate stationary phase deletions in plasmid pMC874

Deletions in the plasmid pMC874 take place in resting cells incubating on McConkey’s or minimal lactose agar and are time rather than generation dependent. These deletions join the km r promoter to a promoterless lac operon giving rise to Lac + papillae on McConkey’s lactose agar, and can occur in the absence of sequence homologies such as direct or inverted repeats. Using this as a selective screen we isolated 31 mutants designated dli (for deletion increase), which enhanced to different extents the frequency of this unusual class of deletions. Six of these were characterized by phenotypic tests and their ability to stimulate other deletion events such as the excision of Tn10 from various chromosomal sites and the loss of cloned fragments between two EcoR1 sites in the gene for chloramphenicol resistance ( cat) of plasmid pBR325. Two of them showed contrasting phenotypes and were studied further: one ( dli1) stimulated Lac + deletions in pMC874 in resting cells but not Tn10 excision from chromosomal locations in log phase cells, and the other one ( dli2) did exactly the reverse, i.e. it enhanced Tn10 excision but not Lac + deletion incidence. Mapping and complementation tests showed that dli1 is a null mutation in recC and was renamed recC2251. This is strong evidence that resting phase deletions in pMC874 are stimulated by the absence of a functional RecBCD enzyme. The dli2 mutation was identified by mapping and phenotypic tests as a mutation in uvrD, the gene for helicase II, and it was tentatively designated uvrD − dli2. These results show that (1) pMC874 is an excellent system to select mutants for genetic functions involved in the generation of resting phase deletions, and (2) there are at least two major deletion pathways in E. coli, one active in resting and the other in actively dividing cells.

Characterization of the uup locus and its role in transposon excisions and tandem repeat deletions in Escherichia coli.

Null mutations in the Escherichia coli uup locus (at 21.8 min) serve to increase the frequency of RecA-independent precise excision of transposable elements such as Tn10 and to reduce the plaque size of bacteriophage Mu (Uup(-) phenotype). By the combined approaches of physical mapping of the mutations, complementation analyses, and protein overexpression from cloned gene fragments, we have demonstrated in this study that the Uup(-) phenotype is the consequence of the absence of expression of the downstream gene (uup) of a two-gene operon, caused either directly by insertions in uup or indirectly by the polar effect of insertions in the upstream gene (ycbY). The promoter for uup was mapped upstream of ycbY by primer extension analysis on cellular RNA, and assays of reporter gene expression indicated that it is a moderately active, constitutive promoter. The uup mutations were also shown to increase, in a RecA-independent manner, the frequencies of nearly precise excision of Tn10 derivatives and of the deletion of one copy of a chromosomal tandem repeat, suggesting the existence of a shared step or intermediate in the pathways of these latter events and that of precise excision. Finally, we found that mutations that increase the frequency of precise excision of Tn10 are divisible into two categories, depending upon whether they did (uup, ssb, polA, and topA) or did not (mutHLS, dam, and uvrD) also increase precise excision frequency of the mini-Tn10 derivatives. It is suggested that the differential response of mini-Tn10 and Tn10 to the second category of mutations is related to the presence, respectively, of perfect and of imperfect terminal inverted repeats in them.

RecBC and RecF recombination pathways and the induced precise excision of Tn10 in Escherichia coli

Mitomycin C (MMC) treatment or mutations in uvrD enhance the frequency of Tn10 precise excision. We have shown previously that several repair–recombination genes, such as recA, ruv and recF are involved in the induced excision process. In this study, we find that other genes belonging to the RecBC and RecF sexual recombination pathways also participate in this process since mutations in recB, sbcB or recO diminish, though to different degrees, the frequency of Tn10 precise excision induced by MMC treatment or by uvrD mutants. Pairwise combinations of some of these mutations were also tested for Tn10 induced precise excision; most of these double mutants showed additive effects in reducing the frequency of the excision process. The results of these studies suggest that recombinational–repair genes, particularly recF, sbcB and recO have different roles in the induced excision of Tn10 than in recombinational mating.

High and low UV-dose responses in SOS-induction of the precise excision of transposons Tn1, Tn5 and Tn10 in Escherichia coli

UV-inducible precise excision of transposons is a specific SOS-mutagenesis process. It deals with the deletion formation which has previously been demonstrated to involve direct or inverted IS-sequences of transposons. The process was used for revisiting the targeted and untargeted SOS-mutability and its relationship to the key genes for SOS-mutagenesis: the recA, lexA and umuDC. The precise excision of transposons Tn5 and Tn10 from the chromosomal insertion sites ade128 and cyc750 is induced in Escherichia coli K-12 and B cells, wild-type for DNA-repair, both by the low doses of UV-light ranging from 0.25 J m −2 to 2.5 J m −2 and the high doses within the range 5.0–40.0 J m −2. Precise excision of these transposons induced by the range of low doses incapable to induce targeted point mutations reveals its mostly untargeted nature. This process for the transposon Tn1 is not induced by UV-light within the range of doses 0.25–2.5 J m −2 while its induction is possible by UV-fluences ranging from 5.0 to 40.0 J m −2. A dose–response of the precise excision of Tn1 is similar to that of the UV-induced reversion of trp UAA point mutation that is targeted by nature and contrasts to the UV-inducible precise excision of Tn5 and Tn10. Both types of UV-inducible precise excision, demonstrated either by Tn1 or Tn5 and Tn10, are eliminated by mutations in the lexA, recA and umuDC genes indispensable for UV-induced SOS-mutability. The palindromic structures different for the transposons Tn1, Tn5 and Tn10 are discussed to be involved and affect the targeted and untargeted precise excision of transposons induced by UV-light.

Involvement of recA and recF in the induced precise excision of Tn 10 in Escherichia coli

It has been shown that the increased frequency of precise excision of Tn 10 observed after UV or mitomycin C (MMC) treatment or with uvrD− mutants is recA-dependent. Previous work has also shown that expression of SOS genes is required for UV- or MMC-induced Tn 10 precise excision. In order to determine if the increased excision of Tn 10 in uvrD− mutants requires only expression of recA, or expression of other SOS genes, or both, we studied the precise excision of Tn 10 in lexA3 (Ind−, SOS non-inducible) and lexA3 recAo98 (operator constitutive recA) mutants. The results of these experiments indicate that the induced excision of Tn 10 in the uvrD− null mutant depends on the expression of recA rather than on any of the other genes repressed by LexA. The effect of a null recF mutation on the excision of Tn 10 in a uvrD− mutant was also investigated and found to abolish the increased frequencies of this process. Similarly, the recF mutation was found to decrease markedly the increased precise excision of Tn 10 induced by MMC in a uvrD+ isogenic strain. These observations indicate that recA and recF are involved in the increased frequencies of Tn10 excision exhibited by uvrD− mutants or after MMC treatment. It remains to be determined whether these two genes participate in these two induction processes in the same biochemical pathways.

Multiple roles for divalent metal ions in DNA transposition: distinct stages of Tn10 transposition have different Mg2+ requirements.

Tn10 transposition takes place by a non-replicative mechanism in which the transposon is excised from donor DNA and integrated into a target site. Mg2+ is an essential cofactor in this reaction. We have examined the Mg2+ requirements at various steps in Tn10 transposition. Results presented here demonstrate that Tn10 excision can occur efficiently at a 16-fold lower Mg2+ concentration than strand transfer and that, at Mg2+ concentrations in the range of 60-fold below the wildt-ype optimum, double strand cleavage events at the two transposon ends are completely uncoupled. These experiments identify specific breakpoints in Tn10 transposition which are sensitive to Mg2+ concentration. Whereas the uncoupling of double strand cleavage events at the two transposon ends most likely reflects the inability of two separate IS10 transposase monomers in the synaptic complex to bind Mg2+, the uncoupling of transposon excision from strand transfer is expected to reflect either a conformational change in the active site or the existence of an Mg2+ binding site which functions specifically in target interactions. We also show that Mn2+ relaxes target specificity in Tn10 transposition and suppresses a class of mutants which are blocked specifically for integration. These observations can be explained by a model in which sequence-specific target site binding is tightly coupled to a conformational change in the synaptic complex which is required for catalysis of strand transfer.

Ruv and recG genes and the induced precise excision of Tn10 in Escherichia coli

Induction of precise excision of Tn10 by UV or mitomycin C (MMC) is dependent on the expression of the SOS system. Ruv mutants of Escherichia coli, which are defective in DNA repair and recombination, showed diminished frequencies of both spontaneous and UV- or MMC-induced excision of Tn10 inserted in gal. RecG mutants, which are also defective in DNA repair and recombination, showed decreased induction of Tn10 excision with MMC, but not after UV treatment. A recG ruv double mutant showed a greater decrease in induction of excision with MMC than either single mutant. One can speculate that the Ruv proteins, which are known to be involved in the resolution of Holliday junctions, might also be involved in the resolution of putative intermediates generated during the precise excision of Tn10. RecG protein, whose function partially overlaps those of Ruv proteins, might also have some role in this process.

Observations on the formation of deletions on monomeric and dimeric plasmids in Escherichia coli.

We have studied the formation of spontaneous mutations on plasmids present in the monomeric and dimeric states in a recF strain of Escherichia coli. Two test systems were employed: (i) the precise excision of Tn5 from the tetA gene of the plasmid pBR322 and (ii) operator constitutive (Oc) mutations on the pBR322-derived plasmid pPY97. The rate of Oc mutations was increased by a factor of three when this plasmid was present in the dimeric state compared to the monomeric state and the Oc phenotype was caused by small deletions in the operator sequence. No apparent mutational hot-spot was found. The rate of Tn5 excision was increased on dimeric compared to monomeric plasmids. Excision from a dimeric plasmid usually resulted in two types of mutant plasmids; a dimeric plasmid, where the Tn5 had excised from one of the plasmid units, and a monomeric parental pBR322. A mechanisms to account for this is suggested. Complementation tests revealed that the increased mutation rate on dimeric plasmids is the result of dimers being mutaphilic per se, rather than the result of a general, trans-acting increase in mutation rates of the host, induced by the presence of the dimeric plasmid. Furthermore, it was found that the rate of Tn5 excision from plasmids in the monomeric state was increased when the region carrying the inserted Tn5 was duplicated.

Multiple pathways of deletion formation in Escherichia coli

We are investigating the mechanisms for deletion formation through the use of mutants which alter deletion frequency together with well characterized systems for deletion detection. We report here on three mutations which were isolated for their ability to stimulate deletions in plasmid pMC874 ( dli mutations). The mutation rec-2251 (formerly known as dli1) is a new allele of recBCD, a group of genes coding for the polypeptide components of the major recombination enzyme complex in E. coli; the second one, dli2 may be a new allele of uvrD, which codes for DNA helicase II; and the third one, dli3, has the phenotype of a mismatch repair mutation. Here we compare the effects of mutations in SOS-repair genes to those of the dli mutations on three different deletion events: (a) the deletion of short (60–100-bp) palindromic and non-palindromic inserts in derivatives of plasmid pBR325; (b) larger (600–800-bp) deletions in plasmid pMC874; and (c) the excision of the Tn10 transposon from chromosomal sites. Our results indicate that some form of SOS processing stimulates the loss of palindromes but not non-palindromes in plasmid pBR325 derivatives, and that RecA is necessary for UV-induced excision of Tn10 but this event is inhibited by UmuCD or its homolog MucAB. Each of the dli mutations showed unique effects on different classes of deletions. Mutation rec-2251 stimulated specifically deletions in pMC874 but had no effect on the deletion of non-palindromes in pBR325, and reduced the incidence of the other deletion events tested including loss of palindromic inserts in pBR325 as well as Tn10 excision. Mutation dli2, on the other hand, stimulated all deletions tested to varying extents, while dli3 did not affect markedly deletion formation in pBR325 plasmids but had a large stimulatory effect on both deletions in plasmid pMC874 and Tn10 excision. These results reveal that (a) some SOS-repair functions participate in deletion formation, (b) mutations selected for altering the incidence of one class of deletions may have totally different effects on other deletion events, and (c) the differences in mutant behavior may result in part from the ability of some pathways to discriminate among different deletion intermediates such as hairpins or cruciforms formed by palindromic sequences vs. transient seconday structures stabilized by direct repeats flanking non-palindromic sequences.

Effect of mutations in SOS genes on UV-induced precise excision of Tn10 in Escherichia coli

UV treatment increases the frequency of Tn10 precise excision from different sites of the Escherichia coli chromosome. UV induction of Tn10 excision is not evidenced in a lexA3 (ind−) mutant carrying either a recA+ or a recA730 allele. High levels of RecA synthesized by a recA+ gene not repressible by LexA do not relieve the non-inducibility of Tn10 excision in a lexA3 (ind−) background. This indicates that the expression of an SOS gene different from recA is necessary for the induction of Tn10 excision. In contrast to UV induction of point mutations, this induction does not depend on a functional umuC gene since umuC::Tn5 mutants show increased levels of Tn10 excision from leu, thr or gal after irradiation. MucAB+ plasmid pKM101 which renders cells more UV-mutable for point mutations decreases UV-induced Tn10 excision. These results show that UV-induced Tn10 precise excision requires SOS induction and that it involves a pathway different from point mutagenesis.

Excision of Tn10 from the donor site during transposition occurs by flush double-strand cleavages at the transposon termini.

Tn10 transposition is accomplished without extensive replication of the transposon sequences. Replicative cointegrate formation is precluded by efficient separation of transposon sequences from flanking donor DNA at an early stage in the transposition reaction. We report here that excision of Tn10 from its donor site occurs by a pair of flush double-strand breaks. Breaks occur at each end of the element precisely between the terminal base pair of the element and the first base pair of flanking DNA. This observation provides definitive evidence that cleavage of both strands of the element occurs under the direct control of Tn10 transposase protein. It is highly likely that transposase itself is directly responsible for these cleavages. The implications of this possibility are discussed.

Transposon Tn5 excision in yeast: influence of DNA polymerases alpha, delta, and epsilon and repair genes.

Interaction between short repeats may be a source of genomic rearrangements and deletions. We investigated possible interactions between short (9 base pairs) direct repeats in yeast by using our previously described system for analyzing bacterial transposon Tn5 excision in yeast. Mutations of either POL3 or POL1, the proposed structural genes for polymerases delta and alpha, respectively, yield high levels of excision at semipermissive temperatures. pol2 (corresponding to polymerase epsilon) and pol2 pol3 double mutants do not exhibit enhanced excision. A majority of excision events involve direct repeats and are precise; the remaining imprecise excisions occur within or in the vicinity of the repeats. The three DNA repair pathways identified by rad1, rad6 and rad18, rad50 and rad52 mutations were examined for their possible role in Tn5 excision; no enhancement was observed in mutants. However, the pol3-stimulated Tn5 excision was reduced in rad52 and rad50 mutants. This suggests the potential for interaction between the systems for DNA double-strand break/recombinational repair and DNA synthesis. Based on the suggestion of Morrison et al. [Morrison, A., Araki, H., Clark, A. B., Hamatake, R. H. & Sugino, A. (1990) Cell 62, 1143-1151] that polymerases delta and alpha are responsible for lagging-strand synthesis and that polymerase epsilon is responsible for leading-strand synthesis, we suggest that Tn5 excision is stimulated under conditions of altered lagging-strand synthesis, possibly due to extended opportunities for single-strand interactions between the inverted insertion sequence I550 repeats of Tn5.

RecA-dependent increased precise excision of Tn10 in Salmonella typhimurium

UV irradiation induced the precise excision of Tn10 inserted in met, trp or srl in a Salmonella tryphimurium strain; mitomycin C was also found to induced the frequency of precise excision of Tn10 from srl or met.Precise excision of Tn10 was not increased by either UV or mitomycin C in a recA mutant. Similarly, a recA mutant derived from uvrD strain showed a drastic reduction in the high spontaneous levels of precise excision of Tn10 of this strain. These results indicate that recA is involved in the increased precise excision of Tn10.In contrast to point mutations excision of Tn10 was found of Tn10 was found to be UV inducible in a top mutant.

Yeast mutants with increased bacterial transposon Tn5 excision

Five complementing recessive mutations that exhibit increased bacterial transposon Tn5 precise excision in yeast Saccharomyces cerevisiae were obtained by ethylmethanesulfonate treatment. One of these mutations (tex1) was submitted to extensive genetic analysis. tex1 is a recessive temperature-sensitive mutation resulting in a 20-100-fold increase in Tn5 excision. It also has increased frequencies of ochre mutation reversion, of forward mutation to canavanine resistance, and loss of chromosome III or its right arm. The possible mechanism of tex1 effects is discussed.

Precise excision and instability of the transposon Tn5 in Pseudomonas aeruginosa

Excision of the transposon Tn5 from sites of insertion in plasmid DNA was shown to occur at high frequency in Pseudomonas aeruginosa. Plasmids with Tn5 insertions conjugated poorly into P. aeruginosa and adversely affected growth compared to the respective parental plasmids. The kanamycin-resistance phenotype of Tn5 was expressed poorly in P. aeruginosa and kanamycin-sensitive strains were common during the manipulation of the P. aeruginosa transconjugants. Examination of plasmid DNA isolated from kanamycin-sensitive P. aeruginosa transconjugants revealed excision of Tn5 sequences. A plasmid containing a selectable marker (mercury resistance) inactivated by a Tn5 insertion was constructed, and Tn5 excised precisely, permitting the expression of the mercury-resistance marker at high frequency (10(-3) in P. aeruginosa and at the expected low frequency (10(-7] in Escherichia coli. The recombinational mechanism that promotes frequent Tn5 excision in P. aeruginosa operated in the absence of the P. aeruginosa recA gene product. Fragments of Tn5 were also examined for excision and instability in P. aeruginosa. A plasmid containing the terminal 485 bp of inverted repeat sequences from Tn5, but lacking the transposase or kanamycin-resistance genes, also showed precise excision of Tn5 DNA at high frequency (10(-2] in P. aeruginosa. Unlike plasmids containing a complete Tn5 insertion, this plasmid transferred to P. aeruginosa at normal frequencies and growth of the host was not severely impaired. In contrast, plasmids containing either IS50 element transferred to P. aeruginosa at greatly reduced frequencies, and transconjugants containing the IS50R element (which contains the active transposase gene) were small and especially difficult to maintain. P. aeruginosa transconjugants harbouring a plasmid containing only the DNA between the IS50 elements (which included the kanamycin-resistance gene) were of normal size and stably maintained.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of plasmid pKM101 on umuDC gene function enhancing precise excision of transposons

Mutation in the thr gene with the highest UV-induced reversion frequency has been chosen from a number of mutations arisen from insertion of transposon Tn10 into the umuDC − Salmonella typhimurium. This mutation has been transfered by transduction into the Salmonella strain carrying E. coli umuDC + genes. umuDC − and umuDC + Salmonella strains, bearing the same insertion mutation, were used to study the effec of umuDC genes and of plasmid pKM101 on spontaneous and UV-induced precise excision of Tn10. This process, like point mutagenesis, is shown ro be umuDC-stimulated. Plasmid pKM101 eliminates the effect of umuDC + genes on UV-induced Tn10 precise excision, in contrast to its effect on point mutagenesis, and does not enhance spontaneous precise excision of Tn10.