DNA base sequence changes induced by bromouracil mutagenesis of lambda phage

The base sequence changes induced by bromouracil mutagenesis in the cI gene of phage lambda have been determined by direct sequence analysis. Phage DNA mutagenized during prophage replication or during phage lytic growth showed predominantly A · T → G · C transitions. The frequency of this mutation was strongly sequence-dependent: 5′ A-C-G-C 3′ > A-C(A.C or T) > A(A.G or T). The difference in mutability of bases in the gene is not the result of specificity in mutL-dependent mismatch repair, since phage grown in mutL host cells showed the same distribution of bromouracil mutations. The observations made in phage mutagenized with bromouracil in the prophage state should be representative of bromouracil mutagenesis in the Escherichia coli chromosome.     

Effect of photoreactivation on mutagenesis of lambda phage by ultraviolet light

There is disagreement in the literature as to whether the major mutagenic photoproduct induced in DNA by ultraviolet light is the cyclobutane dipyrimidine dimer, the most common product, or the [6-4] photoproduct, the next most frequent. In the experiments reported here, cyclobutane dimers were removed from irradiated lambda phage DNA by enzymatic photoreactivation, a process thought to affect no other photoproduct. Photoreactivation of lambda phage in host cells and of lambda DNA in solution reduced clear plaque mutants per plaque-forming unit by two-thirds, in host cells with a constant and near-maximal expression of the SOS functions required for mutagenesis. This result is interpreted to mean that removal of cyclobutane dimers in or near the mutated gene reduces mutation induced by ultraviolet light by two-thirds; therefore, cyclobutane dimers in the phage DNA are responsible for most observed mutations. DNA sequences of mutations in photoreactivated phage showed a smaller fraction of G.C to A.T transitions and a larger fraction of A.T to G.C transitions, compared to phage that were not photoreactivated. This suggests that cyclobutane dimers at TC and CC sites are particularly mutagenic.     

Pyrimidine dimers are not the principal pre-mutagenic lesions induced in lambda phage DNA by ultraviolet light

Experiments were performed to examine the role of cyclobutyl pyrimidine dimers in the process of mutagenesis by ultraviolet (u.v.) light. Lambda phage DNA was irradiated with u.v. and then incubated with an Escherichia coli photoreactivating enzyme, which monomerizes cyclobutyl pyrimidine dimers upon exposure to visible light. The photoreactivated DNA was packaged into lambda phage particles, which were used to infect E. coli uvr- host cells that had been induced for SOS functions by ultraviolet irradiation. Photoreactivation removed most toxic lesions from irradiated phage, but did not change the frequency of induction of mutations to the clear-plaque phenotype. This implies that cyclobutyl pyrimidine dimers can be lethal, but usually do not serve as sites of mutations in the phage. The DNA sequences of mutants derived from photoreactivated DNA showed that almost two-thirds (16/28) were transitions, the same fraction found for u.v. mutagenesis without photoreactivation. These results show that in this system, the lesion inducing transitions (the major type of u.v.-induced mutation) is not the cyclobutyl pyrimidine dimer; a strong candidate for a mutagenic lesion is the Pyr(6-4)Pyo photoproduct. On the other hand, photoreactivation of SOS-induced host cells before infection with u.v.-irradiated phage reduced mutagenesis substantially. In this case, photoreversal of cyclobutyl dimers serves to reduce expression of the SOS functions that are required in the process of targeted u.v. mutagenesis.     

DNA base sequence changes induced by ultraviolet light mutagenesis of a gene on a chromosome in Chinese hamster ovary cells

The DNA base sequence changes induced by mutagenesis with ultraviolet light have been determined in a gene on a chromosome of cultured Chinese hamster ovary (CHO) cells. The gene was the Escherichia coli gpt gene, of which a single copy was stably incorporated and expressed in the CHO cell genome. The cells were irradiated with ultraviolet light and gpt- colonies were selected by resistance to 6-thioguanine. The gpt gene was amplified from chromosomal DNA by use of the polymerase chain reaction (PCR), and the amplified DNA sequenced directly by the dideoxy method. Of the 58 sequenced mutants of independent origin 53 were base change mutations. Forty-one base substitutions were single base changes, ten had two adjacent (or tandem) base changes, and one had two base changes separated by a single base-pair. Only one mutant had a multiple base change mutation with two or more well separated base changes. In contrast much higher levels of such mutations were reported in ultraviolet mutagenesis of genes on a shuttle vector in primate cells. Two deletions of a single base-pair were observed and three deletions ranging from 6 to 37 base-pairs. The mutation spectrum in the gpt gene had similarities to the ultraviolet mutation spectra for several genes in prokaryotes, which suggests similarities in mutational mechanisms in prokaryotes and eukaryotes.     

Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light

Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst. From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication. A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells. In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced. We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.     

DNA mismatch repair mediates protection from mutagenesis induced by short-wave ultraviolet light

To investigate involvement of DNA mismatch repair in the response to short-wave ultraviolet (UVC) light, we compared UVC-induced mutant frequencies and mutational spectra at the Hprt gene between wild type and mismatch-repair-deficient mouse embryonic stem (ES) cells. Whereas mismatch repair gene status did not significantly affect survival of these cells after UVC irradiation, UVC induced substantially more mutations in ES cells that lack the MutSalpha mismatch-recognizing heterodimer than in wild type ES cells. The global UVC-induced mutational spectra at Hprt and the distribution of most spectral mutational hotspots were found to be similar in mismatch-repair-deficient and wild type cells. However, at one predominant spectral hot spot for mutagenesis in wild type cells, the UVC-induced mutation frequency was not affected by the mismatch repair status. Together these data reveal a major role of mismatch repair in controlling mutagenesis induced by UVC light and may suggest the sequence context-dependent direct mismatch repair of misincorporations opposite UVC-induced pyrimidine dimers.     

Damages induced in lambda phage DNA by enzyme-generated triplet acetone

Exposure of lambda phage to triplet acetone, generated via the oxidation of isobutanal by peroxidase, leads to genome lesions. The majority of these lesions are detected as DNA single-strand breaks only under alkaline conditions, and so true breaks do not occur. Also, no sites sensitive to UV-endonuclease from Micrococcus luteus were found in DNA from treated phage. The participation of triplet acetone in the generation of such DNA damage is discussed.     

Effect of umuC mutations on targeted and untargeted ultraviolet mutagenesis in bacteriophage lambda

Mutagenesis of phage lambda towards clear-plaque phenotype (c+----c) results in two classes of mutants that can be distinguished genetically and morphologically. Indirect mutagenesis, i.e. mutagenesis of unirradiated phage lambda c+ stimulated by the ultraviolet irradiation of the Escherichia coli host, results in mixed bursts (c/c+) of turbid wild-type and clear-plaque mutant phages. Pure bursts of lambda c mutants are induced by irradiation of the phage genome. Irradiation of both phages and host bacteria stimulates the production of the two classes of mutant clones. We show that three different mutant alleles of the E. coli umuC gene only prevent the appearance of pure bursts of clear-plaque mutants, while mixed bursts are produced at least as frequently in umuC mutants as in the umuC+ parent.     

Inactivation and mutagenesis of lambda phage by O-methylhydroxylamine and O-delta-aminooxybutylhydroxylamine

The inactivation and the mutagenesis of lambda phage Cl 857 virR by O-methylhydroxylamine (OMHA) and O-delta-aminooxybuthylhydroxylamine (delta-HA) were studied. The inactivation of OMHA-treated phage was shown to be stronger in E. coli polA cells defective in DNA-polymerase I as compared to wild-type host E. coli W3350. In contrast delta-HA caused similar phage inactivation in these two strains. Wave-type kinetics of the inactivation and the mutagenesis of phage by OMHA and delta-HA was observed. delta-HA appeared to be a more effective mutagen than OMHA: it induced higher mutant yield at a given level of inactivation.     

Mutagenesis of lambda phage: Weigle mutagenesis is induced by coincident lesions in the double helical DNA of the host cell genome

Summary We have studied the increase in mutation in mutagenized lambda phage when the host cells are also irradiated with ultraviolet light, Weigle mutagenesis. The increase in mutation is induced mainly by coincidences between a radiation-produced lesion in one strand of the host cell DNA and a second lesion in the complementary strand. This conclusion is based on experiments in which incorporation of the base analog bromouracil sensitized the host cells to ultraviolet light. For the same number of bromouracils incorporated per cell, uniform substitution gave a higher level of Weigle mutagenesis than did substitution in only one strand of the DNA double helix. The data also show some induction of Weigle mutagenesis by processes linear in ultraviolet fluence; possibilities include: lesions involving both complementary strands such as crosslinks, lesions in one strand opposite pre-existing discontinuities in the complementary strand, and very small contributions to induction from lesions in one strand only of the DNA.     

N-terminal sequence of phage lambda repressor

Summary Lambda repressor was purified from an E. coli strain which produces 150 times more lambda repressor than a single lysogen. The sequence of the fifty N-terminal residues was determined by automated Edman degradation. It contains 43% of all arginine and lysine residues of the chain and constitutes according to the genetic data of Oppenheim et al. (1975) a substantial part of the operator-DNA-binding site of the repressor.     

Amplification of the phage lambda DNA sequence by polymerase chain reaction using thermostable DNA polymerase]

The efficiency of phage DNA amplification by the method of polymerase chain reaction (PCR) with Tth DNA-polymerase was studied for optimization of PCR conditions. The effect on amplification efficiency of medium ionic strength and pH, the presence of univalent cations, detergents, gelatin, ATP, pyrophosphate, SH-reagents and ratio of concentrations of Mg and dNTPs, primers and template was studied. It has been found that a pH optimum for PCR with Tth DNA-polymerase varies from 8.5 to 9.0. An ionic strength optimum for PCR is about 0.08. The influence of univalent cations on the activity of Tth DNA-polymerase can be expressed as NH4+ greater than Na+ greater than K+. 0.01% Tween-20 significantly increases the efficiency of PCR and 0.01% gelatin inhibits it. Addition of ATP, pyrophosphate, SH-reagents to the reaction mixture did not increase the yield of PCR product. It has been also shown that for the given PCR-system an optimum Mg/dNTPs molar ratio is within the range of 1.5-2.0. An optimum concentration of each of the pair of primers for this PCR-system is about 0.3 microM. The possibility of PCR-amplification of 500-8500 b.p. DNA fragments has been demonstrated.     

Damaged-site independent mutagenesis of phage lambda produced by inducible error-prone repair

The existence of damaged-site independent mutagenesis is confirmed here by scoring the appearance of clear-plaque (c-) or virulent (vir) forward mutations on intact (non-irradiated) phage lambda grown on UV-irradiated E. coli K12 hosts. The mutation frequency was measured as a function of the incubation time between the occurrence of host DNA lesions and phage infection. The time course of mutagenesis of intact phage followed the induction pattern observed upon UV-reactivation of UV-damaged phage by Defais et al. (1976). Intact phage did not mutate in UV-irradiated hosts carrying the uvm-25 mutation known to prevent the occurrence of UV-reactivation. These findings suggest that damaged-site independent mutagenesis results from inducible error-prone repair. Clear-plaque mutations arising on intact phage were mostly found in phage bursts consisting of clear and turbid plaque formers whereas UV-damaged phage gave rise to mostly clear-plaque formers. Contrarily to damaged-site dependent mutagenesis, damaged-site independent mutagenesis can arise even at late times during the phage replication cycle. Our data indicate that about half of the phage mutations that arise upon UV-reactivation are damaged-site independent mutations. Replication of intact phage DNA in a host during induction of SOS functions provides a sensitive assay for the detection of damaged-site independent mutagenesis.     

Isolation of lambda phage DNA by hydroxylapatite chromatography

A simple and rapid (1 day) method for preparation of lambda phage DNA was proposed. The method included two main steps: (a) growth and lysis of bacteria containing lambda phage and (b) purification of lambda phage DNA by hydroxylapatite chromatography. The phage DNA prepared by this method was intact and free of RNA, proteins, and bacterial DNA.     

Unusual base sequence arrangement in phage phi 29 DNA.

Susceptibility of Bacillus subtilis phage phi 29 DNA to 34 different restriction endoculceases was determined. Three enzymes, BglI, XbaI and BstEII, were found to cleave phi 29 DNA only once at specific sites. The sites of these single cleavages have been mapped. Thirteen enzymes did not cut phi 29 DNA. phi 29 HindIII DNA fragments inserted into pBR313 plasmid and propagated in Escherichia coli, were resistant to these restriction endonucleases. This result suggests that the insusceptibility is due to the absence of the nucleotide sequences on phi 29 recognized by the enzymes, and not to the presence of modified nucleotides.     

Mutagenesis and mutation transfer induced by ultraviolet light in plasmid-cloned DNA

We describe here simple techniques for increasing the frequency of UV-induced mutations in a DNA fragment cloned in plasmid pBR322. Irradiation of both the host and the plasmid DNA before transformation is necessary to produce new mutations in the plasmid DNA, presumably because the UV-damaged pBR322 replicon cannot efficiently induce the error-prone repair pathway of Escherichia coli. In contrast, U V irradiation of the plasmid DNA alone before transformation primarily causes the transfer of preexisting mutations from the host chromosome to homologous DNA present in the plasmid. The only other kind of mutants obtained were large deletions of the plasmid DNA. Two chromosomal mutations from the host galK gene and one from the lacZ gene have been transferred to the plasmid by UV irradiation of the plasmid DNA alone. The technique can thus be of general use.     

Timing of ultraviolet light induced mutagenesis in simian virus 40 relative to viral DNA replication in monkey cells

Summary Simian virus 40 (SV40) was used to probe ultraviolet light (UV) — induced mutation in mammalian cells. Viral mutations were scored as reversions of early and late temperature-sensitive (ts) mutants to the wild-type (WT) phenotype. When virus was exposed to moderate or high UV doses, WT revertants were obtained at a frequency related to the square of the dose from two early (tsA) and one late (tsBC) mutant grown at the restrictive temperature. The reversions generated in the progeny of UV-irradiated early mutants presumably arose before the onset of viral DNA replication because, at the non-permissive temperature, tsA mutants are unable to express the functions responsible for the initiation of viral DNA synthesis. Moreover, the early mutant tsA209 underwent similar levels of induced reversion at the permissive and restrictive temperatures, suggesting that the pre-replicative mutational pathway might predominate for moderately and heavily irradiated virus, even under conditions where DNA synthesis can be initiated. The analysis of bursts from revertant plaques produced at the restrictive temperature was consistent with this interpretation. Although the mechanism of pre-replicative mutagenesis is not known, it is likely to be mediated by cellular activities owing to the low genetic complexity of the virus.