Mutants of Escherichia Coli with Increased Fidelity of DNA Replication

SINEs are short interspersed repeated DNA elements which are considered to spread throughout genomes via RNA intermediates. Polymorphisms with regard to the presence or absence of SINE are occasionally observed in a specific location of a genome. We modeled the evolution of SINEs with regard to this type of polymorphism. Because SINEs are rarely deleted, multiplication of elements is confined to a certain period, and a few master copies are considered to be responsible for their multiplication, the usual population genetic models of transposable elements assuming the equilibrium state are not applicable to describe the evolution of SINEs. Taking into account these properties and assuming selective neutrality, we computed conditional probabilities of finding a SINE at a specific site given that this site is first found because it is occupied by a SINE in an original sample. Using these probabilities, we investigated ways to estimate the multiplication period and infer relationships among populations. The latter inference procedures are shown to be strongly dependent on the multiplication period.     

Isolation and Characterization of Escherichia Coli Mutants with Altered Rates of Deletion Formation

Using site-specific mutagenesis in vitro we constructed a genetic system to detect mutants with altered rates of deletion formation between short repeated sequences in Escherichia coli. After in vivo mutagenesis with chemical mutagens and transposons, the system allowed the identification of mutants with either increased or decreased deletion frequencies. One mutational locus, termed mutR, that results in an increase in deletion formation, was studied in detail. The mutR gene maps at 38.5 min on the E. coli genetic map. Since the precise excision of many transposable elements is also mediated at short repeated sequences, we investigated the effects of the mutant alleles, as well as recA, on precise excision of the transposon Tn9. Neither mutR nor recA affect precise excision of the transposon Tn9, from three different insertions in lacI, whereas these alleles do affect other spontaneous deletions in the same system. These results indicate that deletion events leading to precise excision occur principally via a different pathway than other random spontaneous deletions. It is suggested that, whereas precise excision occurs predominantly via a pathway involving replication enzymes (for instance template strand slippage), deletions on an F'factor are stimulated by recombination enzymes.     

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.     

Palindromy and the Location of Deletion Endpoints in Escherichia Coli

The contributions of direct and inverted repeats to deletion formation were studied by characterizing Ampr revertants of plasmids with a series of insertion mutations at a specific site in the pBR322 ampicillin resistance (amp) gene. The inserts at this site are palindromic, variable in length, and bracketed by 9- or 10-bp direct repeats of amp sequence. There is an additional direct repeat composed of 4 bp within the insert and 4 bp of adjoining amp sequence. DNA sequencing and colony hybridization of Ampr revertants showed that they contained either the parental amp sequence, implying deletion endpoints in the flanking 9- or 10-bp repeats, or a specific 1-bp substitution, implying endpoints in the 4-bp repeats. Although generally direct repeats seem to be used as deletion endpoints with a frequency proportional to their lengths, we found that with uninterrupted palindromes longer than 32 bp, the majority of deletions ended in the 4 bp, not the 9- or 10-bp repeats. This preferential use of the shorter direct repeats associated with palindromes is interpreted according to a DNA synthesis-error model in which hairpin structures formed by intrastrand pairing foster the slippage of nascent strands during DNA synthesis.     

On the Deletion of Inverted Repeated DNA in Escherichia Coli: Effects of Length, Thermal Stability, and Cruciform Formation in Vivo

We have studied the deletion of inverted repeats cloned into the EcoRI site within the CAT gene of plasmid pBR325. A cloned inverted repeat constitutes a palindrome that includes both EcoRI sites flanking the insert. In addition, the two EcoRI sites represent direct repeats flanking a region of palindromic symmetry. A current model for deletion between direct repeats involves the formation of DNA secondary structure which may stabilize the misalignment between the direct repeats during DNA replication. Our results are consistent with this model. We have analyzed deletion frequencies for several series of inverted repeats, ranging from 42 to 106 bp, that were designed to form cruciforms at low temperatures and at low superhelical densities. We demonstrate that length, thermal stability of base pairing in the hairpin stem, and ease of cruciform formation affect the frequency of deletion. In general, longer palindromes are less stable than shorter ones. The deletion frequency may be dependent on the thermal stability of base pairing involving approximately 16-20 bp from the base of the hairpin stem. The formation of cruciforms in vivo leads to a significant increase in the deletion frequency. A kinetic model is presented to describe the relationship between the physical-chemical properties of DNA structure and the deletion of inverted repeats in living cells.     

Localization of Ribosome Formation in Escherichia Coli

Abstract

A number of ribonucleoprotein fractions have been isolated from Escherichia coli K 12, one of which is more strongly bound to the cell membrane than the rest and can be detached only by deoxycholate treatment. Several properties have been analyzed. The following are common to all fractions: Sedimentation characteristics of sub-units; Sedimentation constant, nucleotide composition and capacity to hybridize with DNA of RNA; electrophoretic behaviour of proteins. Other properties differ in the various fractions: cell membrane fractions have higher RNA/protein ratio, are more sensitive to RNAase and dissociate more easily into two subunits. By examining the rate of incorporation of [2-¹⁴C]-uracil into the various fractions, it has been found that cell membrane-bound ribonucleoprotein fractions contain a higher proportion of newly formed rRNA and part of this early-labeled rRNA is contained in particles sedimenting as ribosome precursors. Moreover, by isolating ribonucleoprotein fractions from cells grown in the presence of chloramphenicol, cell membrane-bound riconucleoprotein fraction is richer in CM particles. All these results lead to the conclusion that this fraction contains a higher proportion of ribosome precursors. The meaning of these results is discussed.     

Duplication Mutation as an Sos Response in Escherichia Coli: Enhanced Duplication Formation by a Constitutively Activated Reca

The SOS response in Escherichia coli involves the induction of a multioperon regulatory system, which copes with the presence of DNA lesions that interfere with DNA replication. Induction depends on activation of the RecA protein to cleave the LexA repressor of SOS operons. In addition to inducible DNA repair, the SOS system produces a large increase in the frequency of point mutations. To examine the possibility that other types of mutations are induced as part of the SOS response, we have studied the production of tandem duplications. To avoid the complications of indirect effects of the DNA lesions, we have activated the SOS response by a constitutive mutation in the recA gene, recA730. The introduction of the recA730 mutation results in an increase in duplications in the range of tenfold or greater, as judged by two different criteria. Based on its genetic requirements, the pathway for induced duplication formation is distinct from the point mutation pathway and also differs from the major normal recombination pathway. The induction of pathways for both duplications and point mutations shows that the SOS system produces a broad mutagenic response. We have suggested previously that many types of mutations might be induced by severe environmental stress, thereby enhancing genetic variation in an endangered population.     

Local DNA Sequence Control of Deletion Formation in Escherichia coli Plasmid Pbr322

The specificity of deletion formation was studied using tests involving reversion of palindromic insertion mutations. Insertions of a Tn5-related transposon at 13 sites in the ampicillin-resistance (amp) gene of plasmid pBR322 were shortened to a nested set of perfect palindromes, 22, 32 and 90 bp long. We monitored frequencies of reversion to Ampr, which is the result of deletion of the palindrome plus one copy of the flanking 9 bp direct repeats (which had been formed by transposition). Revertant frequencies were found to depend on the location and the sequence of the palindromic insert. Changing a 45-kb interrupted palindrome to a 22-bp perfect palindrome stimulated deletion formation by factors of from fourfold to 545-fold among the 13 sites, while elongation of the perfect palindrome from 22 to 90 bp stimulated deletion formation by factors of from eight- to 18,000-fold. We conclude that deletion formation is strongly affected by subtle features of DNA sequence or conformation, both inside and outside the deleted segment, and that these effects may reflect specific interactions of DNA processing proteins with template DNAs.     

The Influence of Primary and Secondary DNA Structure in Deletion and Duplication between Direct Repeats in Escherichia Coli

We describe a system to measure the frequency of both deletions and duplications between direct repeats. Short 17- and 18-bp palindromic and nonpalindromic DNA sequences were cloned into the EcoRI site within the chloramphenicol acetyltransferase gene of plasmids pBR325 and pJT7. This creates an insert between direct repeated EcoRI sites and results in a chloramphenicol-sensitive phenotype. Selection for chloramphenicol resistance was utilized to select chloramphenicol resistant revertants that included those with precise deletion of the insert from plasmid pBR325 and duplication of the insert in plasmid pJT7. The frequency of deletion or duplication varied more than 500-fold depending on the sequence of the short sequence inserted into the EcoRI site. For the nonpalindromic inserts, multiple internal direct repeats and the length of the direct repeats appear to influence the frequency of deletion. Certain palindromic DNA sequences with the potential to form DNA hairpin structures that might stabilize the misalignment of direct repeats had a high frequency of deletion. Other DNA sequences with the potential to form structures that might destabilize misalignment of direct repeats had a very low frequency of deletion. Duplication mutations occurred at the highest frequency when the DNA between the direct repeats contained no direct or inverted repeats. The presence of inverted repeats dramatically reduced the frequency of duplications. The results support the slippage-misalignment model, suggesting that misalignment occurring during DNA replication leads to deletion and duplication mutations. The results also support the idea that the formation of DNA secondary structures during DNA replication can facilitate and direct specific mutagenic events.     

Double-Stranded Gap Repair of DNA by Gene Conversion in Escherichia Coli

We demonstrated repair of a double-stranded DNA gap through gene conversion by a homologous DNA sequence in Escherichia coli. We made a double-stranded gap in one of the two regions of homology in an inverted orientation on a plasmid DNA molecule and introduced it into an E. coli strain which has the RecE system of recombination (genotype; sbcA23 recB21 recC22). We detected repair products by genetic selection. The repair products were those expected by the double-strand-gap repair model. Gene conversion was frequently accompanied by crossing over of the flanking sequences as in eukaryotes. This double-strand gap repair mechanism can explain plasmid recombination in the absence of an artificial double-stranded break reported in a companion study by Yamamoto et al.     

Mutator Phenotype Induced by Aberrant Replication

We have identified thermosensitive mutants of five Schizosaccharomyces pombe replication proteins that have a mutator phenotype at their semipermissive temperatures. Allele-specific mutants of DNA polymerase δ (polδ) and mutants of Polα, two Polδ subunits, and ligase exhibited increased rates of deletion of sequences flanked by short direct repeats. Deletion of rad2⁺, which encodes a nuclease involved in processing Okazaki fragments, caused an increased rate of duplication of sequences flanked by short direct repeats. The deletion mutation rates of all the thermosensitive replication mutators decreased in a rad2Δ background, suggesting that deletion formation requires Rad2 function. The duplication mutation rate of rad2Δ was also reduced in a thermosensitive polymerase background, but not in a ligase mutator background, which suggests that formation of duplication mutations requires normal DNA polymerization. Thus, although the deletion and duplication mutator phenotypes are distinct, their mutational mechanisms are interdependent. The deletion and duplication replication mutators all exhibited decreased viability in combination with deletion of a checkpoint Rad protein, Rad26. Interestingly, deletion of Cds1, a protein kinase functioning in a checkpoint Rad-mediated reversible S-phase arrest pathway, decreased the viability and exacerbated the mutation rate only in the thermosensitive deletion replication mutators but had no effect on rad2Δ. These findings suggest that aberrant replication caused by allele-specific mutations of these replication proteins can accumulate potentially mutagenic DNA structures. The checkpoint Rad-mediated pathways monitor and signal the aberrant replication in both the deletion and duplication mutators, while Cds1 mediates recovery from aberrant replication and prevents formation of deletion mutations specifically in the thermosensitive deletion replication mutators.     

Context Effects in the Formation of Deletions in Escherichia Coli

We have examined the frequency with which identical deletions are formed in different chromosomal contexts. A panel of six mutant bla genes containing palindrome/direct repeat structures were moved from pBR322 to three locations: at lambda att, at chromosomal lac, and at F'lac. Deletion of the palindromes and one of the direct repeats results in reversion to Ampr. The frequency of deletion for all alleles declines beyond the reduction in copy number when they are moved from the multicopy plasmid environment to a single-copy chromosome. The magnitude of the declines varies in an allele-specific and location-specific manner. Our data support the hypothesis that context can influence the frequency of mutation independent of the immediate DNA sequence.     

DNA Inversions between Short Inverted Repeats in Escherichia Coli

Using site-specific mutagenesis in vitro, we have constructed Escherichia coli strains that allow the detection of the inversion of an 800-bp segment in the lac region. The invertible segment is bounded by inverted repeats of either 12 or 23 bp. Inversions occurring at these inverted repeats will restore the Lac+ phenotype. Inversions can be detected at both short homologies at frequencies ranging from 0.5 x 10(-8) to 1 x 10(-7). These events, which have been verified by DNA sequence analysis, are reduced up to 1000-fold in strains deficient for either RecA, RecB or RecC. They are not reduced in strains deficient in the RecF, J pathway. These results show that the RecB,C,D system can mediate rearrangements at short sequence repeats, and probably plays a major role in cellular rearrangements.     

Deletion Formation between the Two Salmonella Typhimurium Flagellin Genes Encoded on the Mini F Plasmid: Escherichia Coli Ssb Alleles Enhance Deletion Rates and Change Hot-Spot Preference for Deletion Endpoints

Deletion formation between the 5'-mostly homologous sequences and between the 3'-homeologous sequences of the two Salmonella typhimurium flagellin genes was examined using plasmid-based deletion-detection systems in various Escherichia coli genetic backgrounds. Deletions in plasmid pLC103 occur between the 5' sequences, but not between the 3' sequences, in both RecA-independent and RecA-dependent ways. Because the former is predominant, deletion formation in a recA background depends on the length of homologous sequences between the two genes. Deletion rates were enhanced 30- to 50-fold by the mismatch repair defects, mutS, mutL and uvrD, and 250-fold by the ssb-3 allele, but the effect of the mismatch defects was canceled by the ΔrecA allele. Rates of the deletion between the 3' sequences in plasmid pLC107 were enhanced 17- to 130-fold by ssb alleles, but not by other alleles. For deletions in pLC107, 96% of the endpoints in the recA(+) background and 88% in ΔrecA were in the two hot spots of the 60- and 33-nucleotide (nt) homologous sequences, whereas in the ssb-3 background >50% of the endpoints were in four- to 14-nt direct repeats dispersed in the entire 3' sequences. The deletion formation between the homeologous sequences is RecA-independent but depends on the length of consecutive homologies. The mutant ssb allele lowers this dependency and results in the increase in deletion rates. Roles of mutant SSB are discussed with relation to misalignment in replication slippage.     

Roles of Ruva, Ruvc and Recg Gene Functions in Normal and DNA Damage-Inducible Replication of the Escherichia Coli Chromosome

Induction of the SOS response in Escherichia coli activates normally repressed DNA replication which is termed inducible stable DNA replication (iSDR). We previously demonstrated that initiation of iSDR requires the products of genes, such as recA, recB and recC, that are involved in the early stages of homologous recombination. By measuring the copy number increase of the origin (oriM1) region on the chromosome, we show, in this study, that initiation of iSDR is stimulated by mutations in the ruvA, ruvC and recG genes which are involved in the late stages of homologous recombination. Continuation of iSDR, on the other hand, is inhibited by these mutations. The results suggest that Holliday recombination intermediates, left on the chromosome due to abortive recombination, arrest replication fork movement. Low levels of iSDR and sfiA (sulA) gene expression were also observed in exponentially growing ruvA, ruvC and recG mutants, suggesting that the SOS response is chronically induced in these mutants. We propose that replication forks are arrested in these mutants, albeit at a low frequency, even under the normal (uninduced) conditions.     

Replication bypass of interstrand cross-link intermediates by Escherichia coli DNA polymerase IV

Repair of interstrand DNA cross-links (ICLs) in Escherichia coli can occur through a combination of nucleotide excision repair (NER) and homologous recombination. However, an alternative mechanism has been proposed in which repair is initiated by NER followed by translesion DNA synthesis (TLS) and completed through another round of NER. Using site-specifically modified oligodeoxynucleotides that serve as a model for potential repair intermediates following incision by E. coli NER proteins, the ability of E. coli DNA polymerases (pol) II and IV to catalyze TLS past N(2)-N(2)-guanine ICLs was determined. No biochemical evidence was found suggesting that pol II could bypass these lesions. In contrast, pol IV could catalyze TLS when the nucleotides that are 5' to the cross-link were removed. The efficiency of TLS was further increased when the nucleotides 3' to the cross-linked site were also removed. The correct nucleotide, C, was preferentially incorporated opposite the lesion. When E. coli cells were transformed with a vector carrying a site-specific N(2)-N(2)-guanine ICL, the transformation efficiency of a pol II-deficient strain was indistinguishable from that of the wild type. However, the ability to replicate the modified vector DNA was nearly abolished in a pol IV-deficient strain. These data strongly suggest that pol IV is responsible for TLS past N(2)-N(2)-guanine ICLs.     

Gene Replacement with Linear DNA Fragments in Wild-Type Escherichia Coli: Enhancement by Chi Sites

During conjugation and transduction of Escherichia coli even numbers of recombinational exchanges are required for replacement of a gene on the circular chromosome. We studied gene replacement using a related method of gene transfer (transformation with 6.5-kb linear DNA fragments) as an experimental model for conjugation and transduction. Two properly situated Chi sites, 5' GCTGGTGG 3', stimulated gene replacement ~50-fold, more than the sum of the stimulation by the individual Chi sites. Gene replacement was dependent on RecA and RecB functions. Similar results were obtained with an alternative experimental model in which linear DNA fragments were generated from phage λ by intracellular EcoRI restriction following infection. Dual Chi site-stimulation of these RecA-, RecB-dependent recombination events thus did not depend upon the mode of delivery of the linear DNA into the cells. A single DNA fragment with two Chi sites was sufficient for gene replacement. These results support a one Chi-one exchange hypothesis (``long chunk' gene replacement), stemming from studies with purified RecBCD enzyme, and argue against models in which Chi converts RecBCD enzyme to a state capable of promoting multiple exchanges on one DNA molecule. These results also provide a method for gene targeting in wild-type E. coli and suggest a method for gene targeting in other organisms.