SOS induction in Escherichia coli by single-stranded DNA of mutant filamentous phage: monitoring by cleavage of LexA repressor.

Infection of Escherichia coli in the presence of chloramphenicol with mutant filamentous phage that are defective in the initiation of minus-strand DNA synthesis induces the SOS response as monitored by cellular LexA levels. This observation demonstrates that single-stranded DNA serves as a primary signal for SOS induction in vivo.     

M13 vectors for selective cloning of sequences specifying initiation of DNA synthesis on single-stranded templates

M13 cloning vectors have been developed for the selection of DNA sequences capable of directing initiation of DNA synthesis on single-stranded templates. These vectors are derived from viable M13 mutants containing large deletions in the region of the complementary strand origin. The deletion mutants are defective in the conversion of viral single strands to the duplex replicative form (SS leads to RF) both in vivo and in vitro, give a reduced phage yield and form turbid plaques. A receptor site for foreign single strand initiation determinants has been introduced into the mutants by the insertion of EcoRI linker sequences at the deletion sites. Specific cloned sequences from bacteriophage G4 RF and from Co1E1 DNA restore a clear plaque type and normal phage growth. Selection of clear-plaque isolates obtained by transfection with RF from one of these vectors, M13 delta E101, carrying inserted Co1E1 HaeIII fragments resulted in the selective cloning of one specific fragment, the HaeIII-E fragment. Insertion of either the H or L strand of the HaeIII-E fragment into the M13 delta E101 viral strand gives a clear plaque phenotype, indicating the presence of initiation determinants on both the H- and L-strands of the Co1E1 HaeIII-E fragment. These cloning vectors provide a new means for the functional dissection of replication origins and for the identification of DNA sequences that determine the enzymatic mechanism of discontinuous synthesis along the length of the bacterial chromosome. The ability to assess initiation capability on the basis of plaque morphology also provides a means for rapid genetic analysis of initiation determinants.     

Repair promoted by plasmid pKM101 is different from SOS repair.

In E. coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria. UV-induced reactivation per se was less effective. Bacteria with pKM101 showed no alteration in their division cycle. Plasmid pKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair. Plasmid pKM101 protected E. coli bacteria from UV damage but slightly sensitized them to X-ray lesions. Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional. Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased. Plasmid pKM101 repaired potentially lethal DNA lesions, although wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions.     

Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response.

The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N4-ethenocytosine; epsilon C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in delta recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the epsilon C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in delta recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in delta umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recA-independent alternative induction pathway for SOS gene.     

A runaway-replication plasmid pSY343 contains two ssi signals

Taking advantage of the plaque morphology method, we detected two single-stranded initiation (ssi) signals in the plasmid pSY343; one was in the 170-nucleotide (nt) EcoRV-ThaI segment (170P), and the other was in the 93-nt DraI-FnuDII segment (93F), which were designated as ssiA and ssiB, respectively. We cloned the two ssi signals in the filamentous phage vectors M13 delta lac184 and flR199. A conserved 7-nt consensus sequence involved in the n' recognition site for priming DNA initiation on single-stranded (ss) DNA templates (A. Van der Ende, R. Teerstra, H. Van der Avoort, and P.J. Weisbeek, 1983, Nucleic Acids Res. 11, 4957-4975) was found, three copies in 170P and one in 93F. These two ssi signals contain possible stem and loop structures. The 170P overlapped partly with the origin (ori) region of pSY343 and the 93F was away from the ori region. Growth of chimera phages such as M13 delta lac184/ssiA and M13 delta lac184/ssiB was 38- and 71-fold greater, respectively, than that of M13 delta lac184, 8 h after phage infection. The conversion efficiency in vivo of ss to replicative form (RF) DNA of these chimera phages carrying ssiA and ssiB was 1.9- and 2.2-fold greater, respectively, than that of M13 delta lac184, 50 min after infection.     

Weigle reactivation of diploid M13 phage

Summary The limited ability of ultraviolet (UV)-irradiated E. coli cells to W-reactivate UV-irradiated, single-stranded DNA phages fd and M13 was investigated. The kinetics of induction for W-reactivation of UV-irradiated fd phage are different from that for other SOS functions. W-reactivation of UV-irradiated M13 phage was studied using phage particles that contain at least two single-stranded DNA genomes. No effect on the extent of W-reactivation of diploid phage was observed, compared to that of normal haploid phage, indicating that the mechanism of W-reactivation of single-stranded DNA phages does not involve recombination between partially replicated genomes.     

Selective cloning of a DNA single-strand initiation determinant from phi X174 replicative-form DNA.

An M13 phage deletion mutant, M13 delta E101, developed as a vector for selecting DNA sequences that direct DNA strand initiation on a single-stranded template, has been used for cloning restriction enzyme digests of phi X174 replicative-form DNA. Initiation determinants, detected on the basis of clear-plaque formation by the chimeric phage, were found only in restriction fragments containing the unique effector site in phi X174 DNA for the Escherichia coli protein n' dATPase (ATPase). Furthermore, these sequences were functional only when cloned in the orientation in which the phi X174 viral strand was joined to the M13 viral strand. A 181-nucleotide viral strand fragment containing this initiation determinant confers a phi X174-type complementary-strand replication mechanism on M13 chimeras. The chimeric phage is converted to the parental replicative form in vivo by a mechanism resistant to rifampin, a specific inhibitor of the normal RNA polymerase-dependent mechanism of M13. In vitro, the chimeric single-stranded DNA promotes the assembly of a functional multiprotein priming complex, or primosome, identical to that utilized by intact phi X174 viral strand DNA. Chimeric phage containing the sequence complementary to the 181-nucleotide viral strand sequence shows no initiation capability, either in vivo or in vitro.     

Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli.

A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13 delta lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.     

Replication of M13 oriC bacteriophages in Escherichia coli rep mutant is dependent on the cloned Escherichia coli replication origin.

The involvement of the Escherichia coli rep protein in the replication of M13 chimeric deoxyribonucleic acids (DNAs) carrying the E. coli chromosomal DNA replication origin (oriC) has been examined. Previous studies indicate that the cloning of a 3,550-base-pair sequence of chromosomal DNA containing oriC into an M13 vector allows extensive replication of the M13 oriC chimeric DNA in an E. coli rep-3 mutant. We have extended these studies by preparing a 330-base-pair deletion that specifically deletes the oriC sequence in the M13 oriC DNAs, to demonstrate that the replication observed in the rep-3 host is dependent on the cloned origin. Thus, a DNA-unwinding enzyme other than the rep protein may be involved in the strand separation process accompanying replication which initiates at oriC in the M13 oriC chimeric DNAs and in the E. coli chromosome. The rep assay used for assessing the functionality of the cloned oriC is useful for analysis of any rep-independent origin of replication functional in E. coli. A direct selection for a cloned origin of replication is possible in the rep-3 recA56 host. Since the cloned origin is nonessential for propagation of the M13 chimeric phage in a rep+ host, mutations in the cloned origin may be constructed, and the mutant phage may be examined by a simple transductional analysis of the rep-3 recA56 mutant strain.     

Biological properties of imidazole ring-opened N7-methylguanine in M13mp18 phage DNA.

Guanine residues methylated at the N-7 position (7-MeGua) are susceptible to cleavage of the imidazole ring yielding 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7-MeGua). The presence of Fapy-7-MeGua in DNA template causes stops in DNA synthesis in vitro by E. coli DNA polymerase I. The biological consequences of Fapy-7-MeGua lesions for survival and mutagenesis were investigated using single-stranded M13mp18 phage DNA. Fapy-7-MeGua lesions were generated in vitro in phage DNA by dimethylsulfate (DMS) methylation and subsequent ring opening of 7-MeGua by treatment with NaOH (DMS-base). The presence of Fapy-7-MeGua residues in M13 phage DNA correlated with a significant decrease in transfection efficiency and an increase in mutation frequency in the lacZ gene, when transfected into SOS-induced JM105 E.coli cells. Sequencing analysis revealed unexpectedly, that mutation rate at guanine sites was only slightly increased, suggesting that Fapy-7-MeGua was not responsible for the overall increase in the mutagenic frequency of DMS-base treated DNA. In contrast, mutation frequency at adenine sites yielding A----G transitions was the most frequent event, 60-fold increased over DMS induced mutations. These results show that treatment with alkali of methylated single-stranded DNA generates a mutagenic adenine derivative, which mispairs with cytosine in SOS induced bacteria. The results also imply that the Fapy-7-MeGua in E. coli cells is primarily a lethal lesion.     

Oxidative damage in DNA. Lack of mutagenicity by thymine glycol lesions

Thymine glycol (5,6-dihydroxy-5,6-dihydrothymine) is a base damage common to oxidative mutagens and the major stable radiolysis product of thymine in DNA. We assessed the mutagenic potential of thymine glycols in single-stranded bacteriophage DNA during transfection of Escherichia coli wild-type and umuC strains. cis-Thymine glycols were induced in DNA by reaction with the chemical oxidant, osmium tetroxide (OsO4); modification of thymines was quantitated by using anti-thymine glycol antibody. Inactivation of transfecting molecules showed that one lethal hit corresponded to 1.5 to 2.1 thymine glycols per phage DNA in normal cells, whereas conditions of W-reactivation (SOS induction) reversed 60 to 80% of inactivating events. Forward mutations in the lacI and lacZ' (alpha) genes of f1 and M13 hybrid phage DNAs were induced in OsO4-treated DNA in a dose-dependent manner, in both wild-type and umuC cells. Sequence analysis of hybrid phage mutants revealed that mutations occurred preferentially at cytosine sites rather than thymine sites, indicating that thymine glycols were not the principal pre-mutagenic lesions in the single-stranded DNA. A mutagenic specificity for C----T transitions was confirmed by OsO4-induced reversion of mutant lac phage. Pathways for mutagenesis at derivatives of oxidized cytosine are discussed.     

Protection of nonmodified phageλ againstEcoK restriction mediated byrecA protein

A study was conducted to establish whether the EcoK-specific restriction, which is alleviated in E. coli cells after UV induction of the SOS response (Day 1977), is also alleviated under the influence of an increased level of recA protein without induction of other SOS functions. The host cells used were E. coli K-12, strain AB2497, and its derivatives; the nonmodified phage lambda was a mutant b2b5(vir). An increase of the recA protein level was induced using the plasmid pX02, which is a recombinant of pBR322 carrying the recA gene of E. coli. AB2497(pX02) cells were found to exhibit a lower level of restriction than those without plasmid. The results indicate that the recA protein protects phage DNA during the process of restriction. A further factor affecting restriction is the growth phase of the culture of the restricting host: cells in the late stationary phase exhibit lower restriction than those in the exponential phase of growth. By a combination of these two factors (presence of plasmid pX02 and stationary growth phase) one can reduce the restriction of nonmodified phage about 300 times.     

RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis

The Escherichia coli SOS response to DNA damage is modulated by the RecA protein, a recombinase that forms an extended filament on single-stranded DNA and hydrolyzes ATP. The RecA K72R ( recA2201 ) mutation eliminates the ATPase activity of RecA protein. The mutation also limits the capacity of RecA to form long filaments in the presence of ATP. Strains with this mutation do not undergo SOS induction in vivo . We have combined the K72R variant of RecA with another mutation, RecA E38K ( recA730 ). In vitro , the double mutant RecA E38K/K72R ( recA730,2201 ) mimics the K72R mutant protein in that it has no ATPase activity. The double mutant protein will form long extended filaments on ssDNA and facilitate LexA cleavage almost as well as wild-type, and do so in the presence of ATP. Unlike recA K72R, the recA E38K/K72R double mutant promotes SOS induction in vivo after UV treatment. Thus, SOS induction does not require ATP hydrolysis by the RecA protein, but does require formation of extended RecA filaments. The RecA E38K/K72R protein represents an improved reagent for studies of the function of ATP hydrolysis by RecA in vivo and in vitro .     

Jekyll, a family of phage-plasmid shuttle vectors

A series of shuttle vectors has been constructed, which consist of a plasmid carrying a polylinker sequence and an M13 origin integrated into a lambda vector. A short direct repeat flanking the plasmid allows plasmid excision by homologous recombination. Sequences are cloned into unique restriction sites within the plasmid, and can be recovered either in phage or plasmid form, or can be packaged further as single-stranded DNA phage. These vectors therefore combine the efficiency of phage lambda cloning and screening with the ease of handling or analysing plasmid or M13 clones.     

Role of the E. coli umuC gene product in the repair of single-stranded DNA phage

The umuC product of Escherichia coli has been suggested to have a central role in SOS induced error prone replication of DNA (Kato and Shinoura 1977). To investigate this possibility, we examined the effect of umuC mutations on error prone repair of single and double-stranded DNA phages. No Weigle reactivation of M13 phage was detected in a umuC mutant. Reactivation of lambda phage was reduced but still evident. However mutagenesis occurred in both cases. These results suggest that induced error prone replication of phage DNA can occur via umuC dependent (transdimer synthesis) and umuC independent mechanisms.     

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.     

A simple and rapid nucleotide sequencing strategy and its application in analyzing a rice histone 3 gene

An improved rapid method for sequencing a target DNA is described. A new plasmid, pAA-PZ1, which contains the origin of replication from phage M13 and a portion of the Tn9 transposon was constructed. A long fragment of target DNA cloned into this vector is progressively shortened in vivo from one end by transposon-mediated deletions. The plasmids carrying different lengths of target DNA are then made into single-stranded DNA in the same host upon infection with an M13 phage and their sequence is determined using the dideoxynucleotide chain-termination method. This method bypasses the in vitro enzymatic manipulations for progressive deletions and requires no subcloning. Using this strategy, we sequenced 1.3 kb of rice DNA containing a histone 3 gene within three weeks.     

A yeast-Escherichia coli shuttle vector containing the M13 origin of replication

A yeast-Escherichia coli shuttle vector containing the M13 origin of replication has been constructed. This vector allows selection and replication in both Saccharomyces cerevisiae and E. coli, as well as single-stranded packaging from E. coli upon infection with a helper phage. The presence of a polylinker with various unique restriction sites facilitates the cloning of desired genes.     

Changes in the nature of the sensitivity of an E. coli M strain carrying Inc-group R plasmids, to coliphages T3 and T7

After the transfer of prototype plasmids R6K (IncX), R387 (IncK), R27 (IncH1) and T (IncN) to E. coli M nalr the appearance of histidine-dependent mutants (R27, T), histidine-leucine-dependent mutants (R6K), methionine-proline-dependent mutants (R387) was observed among the resulting transconjugates. The mutations of E. coli M nalr R+ cells induced by the introduction of the plasmids were accompanied by the transformation of the cells from the S-form into the R-form. In contrast to the prototrophs E. coli M nalr, the auxotrophs carrying plasmids R6K, R27, T acquired sensitivity to phage T7, and the methionine-proline-dependent mutant became sensitive to phages T and T7. The above-mentioned plasmids rendered E. coli M cells capable of synthetizing the donor pili. But the adsorption of phages T3 and T7 on the auxotrophic cells, both with and without plasmids, occurred due to their interaction with the cell-wall receptors.