Recombinagenic Processing of Uv-Light Photoproducts in Nonreplicating Phage DNA by the Escherichia Coli Methyl-Directed Mismatch Repair System

Nonreplicating lambda phage DNA in homoimmune Escherichia coli lysogens provides a useful model system for study of processes that activate DNA for homologous recombination. We measured recombination by extracting phage DNA from infected cells, using it to transfect recA recipient cells, and scoring the frequency of recombinant infective centers. With unirradiated phage, recombinant frequencies were less than 0.1%. However, recombination could be increased over 300-fold by prior UV irradiation of the phages. The dependence of recombination on UvrA function varied greatly with UV dose. With phage irradiated to 20 J/m2, recombinant frequencies in repressed infections of uvr+ bacteria were one-fifth those in uvrA infections; with phages irradiated to 100 J/m2, frequencies in uvr+ infections were thirty times higher than in uvrA infections. Most UV-stimulated recombination in uvrA infections appeared to depend on the bacterial methyl-directed mismatch-repair system: frequencies were depressed 5-20-fold in uvrA bacteria also lacking MutH, MutL or MutS functions, and recombinant frequencies decreased with increasing GATC-adenine methylation of phage stocks. The biological activity of nonreplicating UV-irradiated phage DNA declined with time after infection of uvrA cells; this decline was photoproduct-dependent, more marked for undermethylated than overmethylated phage DNA, and depended on host MutHLS functions. In uvr+ bacteria, where the UvrABC system provided an alternative, apparently less efficient, route to recombinagenic DNA, UV-stimulated recombinant frequencies were about twice as high in mutH or mutLS as in mut+ cells, in agreement with hyper-rec mut effects previously described by others.     

Repair of nonreplicating UV-irradiated DNA: cooperative dark repair by Escherichia coli uvr and phr functions.

The system previously used to study recombination of nonreplicating UV-irradiated phage lambda DNA was adapted to study UV repair. Irradiated phages infected undamaged homoimmune lysogens. Pyrimidine dimer content (by treatment with Micrococcus luteus UV endonuclease and alkaline sucrose sedimentation) and a biological activity endpoint (infectivity in transfection of uvrB recA recB spheroplasts) were followed. Unless room light was excluded during DNA extraction procedures, photoreactivation (Phr function) was significant. In uvr delta phr bacteria, repair, by both assays, was very low but not zero. Even when light was totally excluded, Phr function appeared to play a role in Uvr-mediated excision repair: both dimer removal and restoration of infectivity were two to five times as efficient in uvr+ phr+ bacteria as in uvr+ delta phr bacteria. Similarly, UV-irradiated phages plated with higher efficiencies on phr+ than delta phr bacteria even under totally dark conditions. In uvr phr+ repressed infections, removal of dimers from nonreplicating DNA did not increase infectivity as much as in uvr+ infections, suggesting a requirement for repair of nondimer photoproducts by the uvrABC system.     

Isolation of insertion, deletion, and nonsense mutations of the uracil-DNA glycosylase (ung) gene of Escherichia coli K-12.

Two uracil-DNA glycosylase (ung) mutation selection procedures based upon the ability of uracil glycosylase to degrade the chromosomes of organisms containing uracil-DNA were devised to obtain a collection of well-defined ung alleles. In an enrichment procedure, lysogens were selected from Escherichia coli cultures infected with lambda pKanr phage containing uracil in their DNA. (These uracil-DNA phage were prepared by growth on host cells deficient in both dUTPase and uracil-DNA glycosylase.) The lysogenic Kanr population was enriched for uracil glycosylase-deficient mutants by a factor of 10(4). In a phage suicide selection procedure, lambda pung+ phage were unable to form plaques on dut ung cells containing uracil-DNA in their chromosomes, and all of the progeny were lambda pung-. Deletion, insertion (ung::Mu and ung::Tn10), nonsense, and missense mutants were isolated by using these procedures. Extracts of three insertion mutants contained no detectable enzyme activity. All of the other mutant isolates had less than 1% of the normal uracil glycosylase specific activity. The previously studied ung-1 allele, which was derived by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, produced about 0.02% of the normal amount of uracil glycosylase activity. No significant phenotypic differences between ung-1 and ung::Tn10 alleles were observed. Variations of the lysogen selection procedure may be helpful for isolating other DNA glycosylase mutations in E. coli and other organisms.     

Uracil-DNA glycosylase affects mismatch repair efficiency in transformation and bisulfite-induced mutagenesis in Streptococcus pneumoniae.

The generalized mismatch repair system of Streptococcus pneumoniae (the Hex system) can eliminate base pair mismatches arising in heteroduplex DNA during transformation or by DNA polymerase errors during replication. Mismatch repair is most likely initiated at nicks or gaps. The present work was started to examine the hypothesis that strand discontinuities arising after removal of uracil by uracil DNA-glycosylase (Ung) can be utilised as strand discrimination signals. We show that mismatch repair efficiency is enhanced 3- to 6-fold when using uracil-containing DNA as donor in transformation. In order to assess the contribution of Ung to nascent strand discrimination for postreplication mismatch repair, we developed a positive selection procedure to isolate S. pneumoniae Ung- mutants. We succeeded in isolating Ung- mutants using this procedure based on chromosomal integration of uracil-containing hybrid DNA molecules. Cloning and characterization of the ung gene was achieved. Comparison of spontaneous mutation rates in strains either proficient or deficient in mismatch and/or uracil repair gave no support to the hypothesis that Ung plays a major role in targeting the Hex system to neosynthesized DNA strands. However Ung activity is responsible for the increased efficiency of mismatch repair observed in transformation with uracil-containing DNA. In addition Ung is involved in repair of bisulfite-treated transforming DNA.     

UNG-dependent correction of molecular heteroduplexes of M13 phage DNA in Escherichia coli cells

Correction of heteroduplex DNA obtained by hybridization of uracil-containing single-stranded M13mp18 phage DNA and "mutant" synthetic oligonucleotide with deletion of cytosine in SalGI site was studied in ung+ and ung- E. coli strains. Uracil-containing DNA was prepared after growth of phage in an E. coli strain dut- ung-. The DNA was hybridized with "mutant" oligonucleotide then complementary DNA chain was synthesized by T4 DNA polymerase. Ung+ and ung- E. coli cells were transformed by DNA. In all experiments mutation frequency in ung+ was higher than in ung- cells (approximately 6-fold) and reached 11-50%. Absolute number of mutants was higher in ung+ cells. The results indicate that high level of mutagenesis depends on uracil repair system polarizing the correction of heteroduplex DNA.     

Effects of recB21, recF143, and uvrD152 on recombination in lambda bacteriophage-prophage and Hfr by F- crosses.

The effects of the mutation pairs recB21 recF143 and recB21 uvrD152 on the frequency of genetic recombination were investigated in lambda phage-prophage crosses under homoimmune conditions. To prevent recombinants from being formed by the phage red system, these experiments were performed with phages and prophages carrying red and gam mutations. Both spontaneous and damage-induced recombination was measured, the phages being either undamaged or treated with trimethylpsoralen and 360-nm light to cross-link the phage DNA. Control and damaged phages were allowed to infect lysogenic host cells under conditions in which phage gene expression was repressed and phage DNA replication was blocked by lambda immunity. Although the double mutations recB21 recF143 and recB21 uvrD152 reduced recombination in Hfr by F- crosses to 0.3 to 0.02% of the wild-type controls, the presence of these pairs of mutations in the host lysogens had relatively little effect on the results of the phage-prophage crosses. In the latter system, recB21 recF143 reduced spontaneous and damaged-induced recombination by less than threefold whereas recB21 uvrD152 increased it to three times the wild-type level, the increase being attributable to the uvrD mutation. Evidently, the gene products of recB,C uvrD, and recF wee not needed for lambda phage-prophage recombination under repressed conditions.     

Selection by genetic transformation of a Saccharomyces cerevisiae mutant defective for the nuclear uracil-DNA-glycosylase.

A coliphage M13 chimer containing the Saccharomyces cerevisiae TRP1 gene and ARS1 replication origin (mPY2) was grown on an ung- dut- strain of Escherichia coli. The resulting single-stranded phage DNA had 13% of thymine residues substituted by uracil. This DNA failed to transform a delta trp1 yeast strain to prototrophy. However, when a mutagenized yeast stock was transformed with uracil-containing single-stranded mPY2 DNA, unstable transformants were obtained. After plasmid segregation, about half of these were retransformed at a high frequency by uracil-containing single-stranded mPY2 DNA. In vitro, these mutants were defective for uracil-DNA-glycosylase activity. They were designated ung1. Strains containing the ung1 mutation have an increased sensitivity to sodium bisulfite and sodium nitrite but a wild-type sensitivity to methyl methanesulfonate, UV light, and drugs that cause depletion of the thymidylate pool. They have a moderate mutator phenotype for nuclear but not for mitochondrial genes. A low mitochondrial uracil-DNA-glycosylase activity was demonstrated in the mutant strains.     

Human uracil-DNA glycosylase complements E. coli ung mutants.

We have previously isolated a cDNA encoding a human uracil-DNA glycosylase which is closely related to the bacterial and yeast enzymes. In vitro expression of this cDNA produced a protein with an apparent molecular weight of 34 K in agreement with the size predicted from the sequence data. The in vitro expressed protein exhibited uracil-DNA glycosylase activity. The close resemblance between the human and the bacterial enzyme raised the possibility that the human enzyme may be able to complement E. coli ung mutants. In order to test this hypothesis, the human uracil-DNA glycosylase cDNA was established in a bacterial expression vector. Expression of the human enzyme as a LacZ alpha-humUNG fusion protein was then studied in E. coli ung mutants. E. coli cells lacking uracil-DNA glycosylase activity exhibit a weak mutator phenotype and they are permissive for growth of phages with uracil-containing DNA. Here we show that the expression of human uracil-DNA glycosylase in E. coli can restore the wild type phenotype of ung mutants. These results demonstrate that the evolutionary conservation of the uracil-DNA glycosylase structure is also reflected in the conservation of the mechanism for removal of uracil from DNA.     

Selective inhibition of Escherichia coli recBC activities by plasmid-encoded GamS function of phage lambda

The gam locus of bacteriophage lambda encompasses two coding sequences with the same reading frame and translational stop, one corresponding to an Mr 11646 polypeptide (gamS gene), the other to an Mr 16349 polypeptide (gamL gene). A DNA segment encoding gamS but not gamL was placed under lambda pR promoter control (regulated by the cIts857-coded repressor) on a multicopy plasmid, and an insertion mutation (gamS201) was constructed. Expression of gamS+, but not gamS201, inhibited Escherichia coli RecBC nuclease in vivo; the criteria were inhibition of chromosomal DNA degradation after UV irradiation and plating of T4 gene 2- phages. The recB+ C+ bacteria expressing gamS+ were completely or partially similar to recC- mutants with respect to certain phenotypes: defective plating of phages P1 and P2, ability to plate (in a recA- background) lambda red- gam- phages, reduced resistance to UV irradiation, defective SOS induction, decreased colony-forming ability.     

Regulatory light-chains and scallop myosin. Full dissociation, reversibility and co-operative effects

Lambda duplication phages grown for several rounds on Escherichia coli strains containing arl mutations were recombined at elevated frequencies (3 to 6-fold higher) in subsequent test infections. Enhanced recombination of Arl^? phages (grown on arl bacteria) was demonstrable by assays for altered genetic linkages as well as by the standard assay, which measures the conversion of duplication phages (EDTA-sensitive) to single-copy phages (EDTA-resistant). The accumulated potential for enhanced recombination was lost during subsequent growth of the phages on arl⁺ bacteria. Arl^? phages had the same mutation frequencies, at a variety of loci, as control phages; arl bacteria themselves exhibited normal mutation rates. Arl^? phages had normal plating efficiencies and buoyant densities. DNA extracted from Arl^? phages exhibited the same frequency of strand interruption, the same superhelical density (when circularized in vivo), and the same thermal denaturation profile as DNA from phages grown on arl⁺ bacteria. Recombination of Arl^? phages in the presence of λ repressor was very low, as is the case for normal phages. The recombination frequency of ultraviolet light irradiated (80 J/m²) Arl^? phages was more than twice the sum of the frequencies for unirradiated Arl^? phages and irradiated control phages. Substantially increased recombination of Arl^? phages was observed when either the E. coli RecBC, or RecE (but not RecF) pathway was active.     

A human enzyme that liberates uracil from DNA

An enzyme activity which acts specifically on uracil-containing DNA was found in human placenta and cultured fibroblasts. The enzyme liberates uracil from DNA in the presence of EDTA at pH 7.5. Almost equal levels of the activity were found in normal and xeroderma pigmentosum cell lines (complementation group A).     

Role of uracil-DNA glycosylase in the repair of deaminated cytosine residues of DNA in Escherichia coli.

Uracil-DNA glycosylase, which acts specifically on uracil-containing DNA, was purified 250-fold from an extract of Escherichia coli 1100. The enzyme releases free uracil from DNA, producing alkali-labile apyrimidinic sites in the DNA. The enzyme is active on both native and heat-denatured DNA of phage PBS1, which contains uracil in place of thymine. piX174 DNA which had been treated with bisulfite and then at alkaline pH was susceptible to the action of uracil-DNA glycosylase. Since DNA treated with bisulfite alone was less susceptible to the enzyme, it is likely that the enzyme recognizes deaminated cytosine, namely uracil, but not bisulfite adducts of uracil and cytosine in the treated DNA. DNA treated with nitrite or hydroxylamine was not attacked by the enzyme. Enzyme activity acting on bisulfite-treated DNA was absent from an extract of E. coli mutant BD10 (ung). The mutant exhibited higher sensitivity to bisulfite than did the wild-type strain and was unable to reactivate phage T1 pre-exposed to bisulfite and weak alkali.     

The chemical mutagen dimethyl sulphate induces homologous recombination of plasmid DNA by increasing the binding of RecA protein to duplex DNA.

The role of different DNA damages in the stimulation of homologous recombination was studied by using an in vivo plasmid recombination assay. Dimethyl sulphate (DMS) treatment of plasmid DNA induced a 20-50-fold increase in the frequency of recombinational events. DMS treatment also stimulated RecA protein binding to double-stranded DNA. In contrast, plasmid DNA containing uracil, which, like DMS, is also subject to repair, was less effective in stimulation of recombination. The ability of purified RecA protein to bind DMS-treated or uracil-containing DNA was tested by measuring its ATPase activity. The result indicates that DMS treatment, but not uracil incorporation, stimulates RecA protein binding to DNA. We conclude, that the main reason (or the first step) for stimulation of recombination by mutagens is activation of RecA binding to damaged DNA.     

Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli III. Enhancement of excision repair in UV-treated bacteria

Summary The question of whether induction of the SOS response in Escherichia coli increases the efficiency of excision repair was addressed by measuring repair of UV-damaged nonreplicating lambda phage DNA in previously irradiated bacteria. Prior UV irradiation of lex ⁺ bacteria enhanced both the rate of regeneration of infective phage DNA (about 10-fold) and the rate of cyclobutane dimer removal early in repressed infections. Indirect induction of SOS-regulated repair activities by the nonreplicating irradiated phage DNA itself seemed negligible. Prior bacterial irradiation reduced the frequency of recombination (loss of a tandem chromosomal duplication) of nonreplicating UV-irradiated DNA. In this respect UV-stimulated recombination of nonreplicating DNA differs from RecF-dependent recombination processes that are stimulated by increased SOS expression.Surprisingly, prior UV irradiation of lexA3 bacteria caused a small but reproducible increase in the regeneration of infective phage DNA.     

Synthesis and metabolism of uracil-containing deoxyribonucleic acid in Escherichia coli

Significant amounts of uracil were found in the deoxyribonucleic acids (DNAs) of Escherichia coli mutants deficient in both uracil-DNA glycosylase (ung) and deoxyuridine 5'-triphosphate nucleotidohydrolase (dut) activities, whereas little uracil was found in the DNAs of wild-type cells and cells deficient in only one of these two activities. The amounts of uracil found in the DNAs of dut ung mutants were directly related to the growth temperature of the cultures, apparently because the deoxyuridine 5'-triphosphate nucleotidohydrolase synthesized by dut mutants was temperature sensitive. The dut mutant used failed to grow exponentially, became filamentous at temperatures above 25 degrees C, and exhibited a hyperrec phenotype; however, the ung mutation suppressed all of these effects. Although the dut ung mutants grew exponentially at all temperatures, their growth rates were always slower than the growth rate of the wild type. Since pool size measurements indicated that both deoxyuridine triphosphate and deoxythymidine triphosphate pools were markedly elevated in dut mutants, the reduced growth rate of dut ung cells apparently was due to the actual presence of uracil in the DNA, rather than to a deficiency of deoxyuridine triphosphate and deoxyribosylthymine triphosphate for DNA synthesis. The presence of uracil in E. coli donor DNA also markedly reduced the recombination frequency when the recipient cells were ung+, indicating that DNA repair commenced before the entering DNA could be replicated.     

Specific mutator effects of ung (uracil-DNA glycosylase) mutations in Escherichia coli.

Studies of trpA reversions revealed that G:C leads to A:T transitions were stimulated about 30-fold in E. coli ung mutants, whereas other base substitutions were not affected. A dUTPase (dut) mutation, which increases the incorporation of uracil into DNA in place of thymine, had no significant effect on the rate of G:C leads to A:T transitions. The results support the proposal that the glycosylase functions to reduce the mutation rate in wild-type cells by acting in the repair of DNA cytosine residues that have undergone spontaneous deamination to uracil. Further support was provided by the finding that when lambda bacteriophages were treated with bisulfite, an agent known to produce cytosine deamination, the frequency of clear-plaque mutants was increased an additional 20-fold by growth on an ung host. Bisulfite-induced mutations of the cellular chromosome, however, were about equal in ung+ and ung strains; it was found that during the treatment of ung+ cells with bisulfite, the glycosylase was inactivated.     

Formation of lambda transducing phage in vitro: involvement of DNA gyrase

We found that transducing phages carrying the gal or bio regions of the Escherichia coli genome were formed during in vitro packaging of endogenous lambda DNA. Structural analysis of the transducing phage genomes indicated that they were formed by abnormal excision of lambda prophage. Formation of transducing phages was stimulated by oxolinic acid, an inhibitor of DNA gyrase, implying that DNA gyrase participates in the abnormal excision of lambda prophage. When pBR322 DNA was added to the reaction mixture, transducing phages into which pBR322 had been inserted were produced at a high frequency. This reaction was also stimulated by oxolinic acid. Sequence analyses revealed that pBR322 is inserted into the sites of abnormal excision of the prophage. These results show that transducing phages can be formed by DNA gyrase-dependent illegitimate recombination in an in vitro system and that secondary recombination takes place frequently at the site where the first recombination occurs.     

AP endonuclease and uracil DNA glycosylase activities in Deinococcus radiodurans.

An endonuclease specific for apurinic/apyrimidinic (AP) sites was identified and purified from extracts of Deinococcus radiodurans. The enzyme is 34.5 kD, has no activity towards normal, alkylated, uracil-containing, or UV-irradiated DNA, and is active in the presence of EDTA. The addition of up to 10 mM Mg2+ or Mn2+ did not affect activity, but higher concentrations were inhibitory. There is no associated exonuclease activity, either in the presence or absence of divalent cation. Optimal reaction conditions were 150 mM NaCl and pH 7.5. A uracil DNA glycosylase was also detected, active in the presence of EDTA, selectively removing uracil from DNA without generating other byproducts. The optimal reaction conditions were 50 mM NaCl and pH 7.5. Implications for base excision repair in D. radiodurans are discussed.     

Specialized transduction with lambda plac5: dependence on recA and on configuration of lac and att lambda.

The construction of lambda plac5 transducing phages carrying various lacZ alleles is described. Genetically disabled (N- N- P-) lambda plac transducing the phages were used to study the dependence of specialized transduction on host RecA function and on the location of the lacZ gene in the recipient strain. In the absence of site-specific recombination at att lambda, transduction was completely dependent on host RecA function. Regardless of the configuration of att lambda, lambda plac transducing phages recombined at a 20- to 50-fold higher frequency with F42 lac than with a lac gene located in the cellular chromosome. Deletion mutants of lacZ in the recipient strain were used to show that the probability of lac recombination resulting from lambda plac infection is apparently proportional to the amount of homology between the parental lacZ genes.