Mismatch repair and recombination in E. coli

The involvement of the E. coli methyl-directed and very short patch (vsp) mismatch repair systems in bacteriophage lambda recombination has been studied. Genetic crosses and heteroduplex transfection experiments were performed using lambda phages with sequenced mutations in the cl gene. The results indicate that methyl-directed repair does operate during bacteriophage lambda recombination but generally does not contribute to the formation of recombinants involving close markers. Vsp repair apparently acts during bacteriophage lambda recombination to produce recombinants involving close markers because its action does not involve extensive excision tracts. Marker-specific hyperrecombination and the apparent clustering of genetic exchanges in bacteriophage lambda recombination can be accounted for by the action of the vsp repair system.     

Expansion of DNA repeats in Escherichia coli: effects of recombination and replication functions.

Duplication or expansion of directly repeated sequence elements is associated with a number of human genetic diseases. To study the mechanisms of repeat expansion, we have developed a plasmid assay in Escherichia coli. Our assay involves two simple repeats of 787 bp in length; expansion to three or more copies of the repeat can be selected by restoration of an intact tetracycline-resistance gene. Expansions occurred at relatively high rates, >10(-5), in the population. Both RecA-dependent recombination and RecA-independent slipped misalignments contributed to the observed expansion events. Mutations that impair DNA polymerase III (DnaE, DnaQ subunits) or the replication fork helicase, DnaB, stimulated both RecA-dependent and RecA-independent expansion events. In these respects, the properties of repeat expansion resemble repeat deletion and suggest that difficulties in DNA replication may trigger both classes of rearrangements. About 20% of the RecA-independent expansion events are accompanied by reciprocal sister-chromosome exchange, producing dimeric plasmids carrying one triplicated and one deleted locus. These products are explained by a model involving misaligned strands across the replication fork. This model predicts that the location of a replication stall site may govern the types of resulting rearrangements. The specific location of such a stall site can also, in theory, account for propensity towards expansion or deletion of repeat arrays. This may have relevance to trinucleotide repeat expansion in human genetic disease.     

Mismatch repair in the antimutator Escherichia coli mud

Antimutators are genetic mutants that produce mutations at reduced rates compared to the wild type strain. They are interesting because they may provide insights into the mechanisms by which spontaneous mutations occur. We have investigated a reported antimutator strain of Escherichia coli termed mud for its possible mechanism. The mud strain exhibits a decrease in both spontaneous mutagenesis and mutability with alkylated agents and base analogs. These types of DNA lesions are known to be the substrates for the E. coli methyl-directed mismatch repair encoded by the mutHLSU system. We investigated whether the putative antimutator effect results from the increased expression or activity of the mutHLSU system. To directly measure the mismatch repair capacity of mud cells, we have transfected them with phage lambda heteroduplexes and scored the fraction of mixed (unrepaired) infective centers. This transfection system has been used routinely to assay mismatch repair capacity in E. coli and other organisms. No difference between mud and wild type cells is observed. From the results of the experiments we conclude that the reported antimutator effect of mud does not result from enhanced mismatch repair capacity. This conclusion is consistent with recently published evidence that the mud effect does not represent a real antimutator effect, but is an artifact due to impaired growth of mud cells under certain selective conditions.     

Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes.

Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products.     

2-aminopurine allows interspecies recombination by a reversible inactivation of the Escherichia coli mismatch repair system

2-Aminopurine treatment of Escherichia coli induces a reversible phenotype of DNA mismatch repair deficiency. This transient phenotype results in a 300-fold increase in the frequency of interspecies conjugational recombination with a Salmonella enterica serovar Typhimurium Hfr donor. This method can be used for the generation of biodiversity by allowing recombination between diverged genes and genomes.     

Excision repair functions in Saccharomyces cerevisiae recognize and repair methylation of adenine by the Escherichia coli dam gene.

Unlike the DNA of higher eucaryotes, the DNA of Saccharomyces cerevisiae (bakers' yeast) is not methylated. Introduction of the Escherichia coli dam gene into yeast cells results in methylation of the N-6 position of adenine. The UV excision repair system of yeast cells specifically responds to the methylation, suggesting that it is capable of recognizing modifications which do not lead to major helix distortion. The UV repair functions examined in this report are involved in the incision step of pyrimidine dimer repair. These observations may have relevance to the rearrangements and recombination events observed when yeast or higher eucaryotic cells are transformed or transfected with DNA grown in E. coli.     

Collapse and repair of replication forks in Escherichia coli

Single-strand interruptions in a template DNA are likely to cause collapse of replication forks. We propose a model for the repair of collapsed replication forks in Escherichia coli by the RecBCD recombinational pathway. The model gives reasons for the preferential orientation of Chi sites in the E. coli chromosome and accounts for the hyper-rec phenotype of the strains with increased numbers of single-strand interruptions in their DNA. On the basis of the model we offer schemes for various repeat-mediated recombinational events and discuss a mechanism for quasi-conservative DNA replication explaining the recombinational repair-associated mutagenesis.     

Ring structure of the Escherichia coli DNA-binding protein RdgC associated with recombination and replication fork repair

The DNA-binding protein, RdgC, is associated with recombination and replication fork repair in Escherichia coli and with the virulence-associated, pilin antigenic variation mediated by RecA and other recombination proteins in Neisseria species. We solved the structure of the E. coli protein and refined it to 2.4A. RdgC crystallizes as a dimer with a head-to-head, tail-to-tail organization forming a ring with a 30 A diameter hole at the center. The protein fold is unique and reminiscent of a horseshoe with twin gates closing the open end. The central hole is lined with positively charged residues and provides a highly plausible DNA binding channel consistent with the nonspecific mode of binding detected in vitro and with the ability of RdgC to modulate RecA function in vivo.     

Control of the initiation of DNA replication in Escherichia coli. I. Negative control of initiation.

A transient stimulation of the initiation of DNA replication during inhibition of protein synthesis has been demonstrated in dnaA5 and dnaA46 mutants. This suggests the existence of a negatively acting control protein (not the dnaA product) which decays rapidly or is removed during a block in protein synthesis. The stimulation of initiation is dependent on a previous accumulation of initiation specific proteins, which suggests that in addition to the negative control other control mechanisms are effective.     

Growth rate-dependent control of chromosome replication initiation in Escherichia coli.

The initiation mass, defined as cell mass per origin of deoxyribonucleic acid replication (optical density units at 460 nm of culture/origins per milliliter of culture), reflects the intracellular concentration or activity of a hypothetical factor that controls initiation of chromosome replication in bacteria. In Escherichia coli B/r, the initiation mass was found to increase about twofold with increasing growth rate between 0.6 and 1.6 doublings per h; at higher growth rates it remained essentially constant (measured up to 2.4 doublings per h). A low-thymine-requiring (thyA deoB) derivative of E. coli B/r, strain TJK16, was found to have a 60 to 80% greater initiation mass than B/r which was independent of the replication velocity and not related to the thyA and deoB mutations. It is suggested that TJK16 had acquired, during its isolation, a mutation in a gene affecting the initiation of deoxyribonucleic acid replication. The initiation age was not altered by this mutation, but other parameters, including deoxyribonucleic acid concentration and cell size, were changed in comparison with the B/r parent, as expected from theoretical considerations.     

Nucleoside triphosphate dependence of repair replication in toluenized Escherichia coli

Ultraviolet irradiation of Escherichia coli stimulates non-conservative DNA synthesis in cells rendered permeable to nucleoside triphosphates by treatment with toluene. This synthesis, like semi-conservative replication, proceeds in the presence of millimolar concentrations of ATP. Unlike semi-conservative replication, the ultraviolet-stimulated DNA synthesis can proceed if other nucleoside triphosphates are substituted for ATP. The selective dependence of semi-conservative replication upon ATP has been used to study the repair mode of synthesis in the absence of the semi-conservative mode and to demonstrate the dependence of ultraviolet-stimulated synthesis upon the uvrA gene product. Studies with recB mutants show that the nucleoside triphosphate-dependent ultravioletstimulated DNA synthesis occurs in strains deficient in the RecBC deoxyribonuclease.     

Mismatch repair and the fidelity of genetic recombination

Two modes of mismatch repair are known to operate in bacteria: long-patch mismatch repair and very short patch mismatch repair. Very short patch mismatch repair systems act on a specific mismatch by conserving only one base pair. Therefore, when very short patch mismatch repair acts on heteroduplex recombination intermediates, it hyper-recombines specific markers by creating patchwork sequences, i.e., apparent multiple exchange events, on the repaired strand. Long-patch mismatch repair is antirecombinagenic, apparently by decomposing heteroduplex DNA or aborting its formation whenever well-recognized mismatches are formed by strand exchange between nonidentical parental sequences. It is postulated here that mismatch-stimulated antirecombination by long-patch mismatch repair is a "proofreading" system assuring high fidelity of homologous recombination. This accounts for chromosomal stability in eucaryotes (i.e., the rare occurrence of chromosomal aberrations and mitotic recombination versus the high frequency of precise sister chromatid exchange), suggests a role for diverged repetitive and other noncoding sequences as chromosomal antirecombination elements, and provides a molecular mechanism for speciation without the necessity of geographical separation.     

Enzymatic control of homologous recombination and hyperrecombination in Escherichia coli

The RecA protein is a central homologous recombination enzyme in the bacterial cell. Forming a right-handed filament on ssDNA, RecA provides for a homology search between two DNA molecules and homologous strand exchange. RecA protects the cell from ionizing radiation and UV light and is capable of completing recombination during normal cell growth. Several mutant and natural RecA forms have a higher recombination potential in vitro and in vivo as compared with the wild-type Escherichia coli RecA, causing hyperrecombination. Recombinational hyperactivity of RecA depends to a great extent on the filamentation dynamics and DNA transferase properties, which may depend not only on specific amino acid substitutions in RecA, but also by defects in cell enzymatic machinery, including RecO, RecR, RecF, RecX, DinI, SSB, and PsiB. The functions of these proteins are currently known at the molecular level, while their roles in hyperrecombination are still incompletely understood. An increase in recombination in vivo is not always advantageous for the cell and is therefore limited by various mechanisms. In addition to the limitations imposed by cell enzymatic machinery, genomic rearrangements aimed at inhibiting the expression of hyperactive RecA are fixed through cell generations via selection against hyperrecombination. The mechanisms regulating hyperactive RecA forms in several model systems are considered.     

Mechanism of dnaB protein action. I. Crystallization and properties of dnaB protein, an essential replication protein in Escherichia coli

Purification and crystallization of dnaB protein from Escherichia coli was performed on a large scale by a simple procedure. From 1.5 kg of cells, 520 mg of dnaB protein were obtained in a 58% yield with a purity greater than 99%. The E. coli cells harbor a high copy-number plasmid carrying the dnaB gene and overproduce the enzyme over 200-fold. The subunit molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 50,000. Based on a native Mr = 290,000 and cross-linking studies that yielded six bands, dnaB protein is judged to be a hexamer, confirming the results of Reha-Krantz, L. J., and Hurwitz, J. (1978) J. Biol. Chem. 253, 4043-4050.     

The dnaK gene of Escherichia coli functions in initiation of chromosome replication.

A newly isolated dnaK mutant of Escherichia coli, which contains the mutation dnaK111, has been found to be conditionally defective in initiation of DNA replication. Mutant cells that were transferred to high temperature exhibited residual DNA synthesis before the synthesis stopped completely. Analysis of the DNA synthesized at high temperature by hybridization with probe DNAs for detection of DNA replicated in the origin (oriC) and terminal (terC) regions has revealed that this mutant is unable to initiate a new round of DNA replication at high temperature after termination of the round in progress. The cells exposed to high temperature were subsequently capable of initiating DNA replication at low temperature in a synchronous manner. DNA synthesis of this mutant became temperature resistant upon inactivation of the rnh gene, similar to that of dnaA mutants, although cell growth of the dnaK mutant with the inactive rnh gene remained temperature sensitive. The dnaK mutation prevented DNA synthesis of lambda bacteriophage at high temperature even in the absence of the rnh gene function.     

Synthesis of linear multimers of OriC and pBR322 derivatives in Escherichia coli K-12: role of recombination and replication functions.

Inactivation of RecBCD nuclease (exonuclease V) and SbcB nuclease (exonuclease I) in Escherichia coli K-12 diverts most of plasmid replication activity from circular monomer production to the synthesis of linear multimers. Linear multimer synthesis has been demonstrated in plasmids of diverse origins and copy numbers, including E. coli minichromosomes. The effect of dnaA, dnaB, recF, and recJ mutations on the rate of linear multimer synthesis in sbcB cells after gam inactivation of RecBCD nuclease was investigated. Results are consistent with the hypothesis that homologous recombination, but not activities at the plasmid origin of replication, is involved in initiation of linear multimer synthesis.     

Genetic control of recombination exchange frequency in Escherichia coli K-12.

The frequency of recombination exchanges per unit length of DNA (Freuld) can be estimated by measuring the scale of the genetic map that is the mean statistical distance between two neighboring crossovers. The scales appear to be equal for the alternative pathways of recombination, RecBCD (wild-type cells) or RecF (recBC- sbcB- sbcC- genotypes). The absolute value of the scale depends on specific experimental conditions. recR, recQ, ruv, recJ and recN genes of the RecF pathway of recombination (recBC- sbcBC- cell genotypes) do not appear to be silent in wild-type cells where the RecBCD pathway predominates. On the contrary, these genes are responsible for the Freuld. The list recF504::Kmr greater than recQ61::Tn3 greater than ruv-54 greater than recJ284::Tn10 shows decreasing efficiency in inhibiting recombination exchanges by these mutations. The recN264 mutation gives a small, but opposite effect of increasing the frequency of recombination exchanges. The effect of the recF and recQ mutations appears to be additive, but that is not the case in combinations of ruv-54 with recF504::Kmr or recQ61::Tn3.     

Role of DNA replication and repair in thymineless death in Escherichia coli

Inhibition of DNA replication with hydroxyurea during thymine starvation of Escherichia coli shows that active DNA synthesis is not required for thymineless death (TLD). Hydroxyurea experiments and thymine starvation of lexA3 and uvrA DNA repair mutants rule out unbalanced growth, the SOS response, and nucleotide excision repair as explanations for TLD.