Depurination-induced infidelity of deoxyribonucleic acid synthesis with purified deoxyribonucleic acid replication proteins in vitro

Removal of purine bases from phi X174 single-stranded DNA leads to increased reversion frequency of amber mutations when this DNA is copied in vitro with purified DNA polymerases. This depurination-induced mutagenesis is observed at three different genetic loci and with several different purified enzymes, including Escherichia coli DNA polymerases I and III, avian myeloblastosis virus DNA polymerase, and eukaryotic DNA polymerases alpha, beta, and gamma. The extent of mutagenesis correlates with the estimated frequency of bypass of the lesion and is greatest with inherently inaccurate DNA polymerases which lack proofreading capacity. With E. coli DNA polymerase I, conditions which diminish proofreading result in a 3-5-fold increase in depurination-induced mutagenesis, suggesting a role for proofreading in determining the frequency of bypass of apurinic sites. The addition of E. coli single-stranded DNA-binding protein to polymerase I catalyzed reactions with depurinated DNA had no effect on the extent of mutagenesis. Analysis of wild-type revertants produced during in vitro DNA synthesis by polymerase I or avian myeloblastosis virus DNA polymerase on depurinated phi X174 amber 3 DNA indicates a preference for insertion of dAMP opposite the putative apurinic site at position 587. These results are discussed in relation both to the mutagenic potential of apurinic sites in higher organisms and to studies on error-prone DNA synthesis.     

The nature of the bond between peptide and carrier molecule determines the immunogenicity of the construct.

The influence of the nature of the bond between a peptide and a (lipidic) carrier molecule on the immunogenicity of that construct was investigated. As types of bonds a thioester-, a disulfide-, an amide- and a thioether bond were investigated. As carrier molecules a peptide, an N-palmitoylated peptide or a C(16)-hydrocarbon chain were used. The biostability of the bond between peptide and carrier molecule is thioether > amide > disulfide > thioester. However, the immunogenic potency of the constructs used was found to be thioester > disulfide > amide > thioether. In conclusion, a construct with a bond between peptide and (lipidic) carrier molecule that is more susceptible to biological degradation is more immunogenic when used in a peptide-based vaccine than a bond that is less susceptible to biological degradation.     

The extreme mutator effect of Escherichia coli mutD5 results from saturation of mismatch repair by excessive DNA replication errors.

Escherichia coli mutator mutD5 is the most potent mutator known. The mutD5 mutation resides in the dnaQ gene encoding the proofreading exonuclease of DNA polymerase III holoenzyme. It has recently been shown that the extreme mutability of this strain results, in addition to a proofreading defect, from a defect in mutH, L, S-encoded postreplicational DNA mismatch repair. The following measurements of the mismatch-repair capacity of mutD5 cells demonstrate that this mismatch-repair defect is not structural, but transient. mutD5 cells in early log phase are as deficient in mismatch repair as mutL cells, but they become as proficient as wild-type cells in late log phase. Second, arrest of chromosomal replication in a mutD5-dnaA(Ts) strain at a nonpermissive temperature restores mismatch repair, even from the early log phase of growth. Third, transformation of mutD5 strains with multicopy plasmids expressing the mutH or mutL gene restores mismatch repair, even in rapidly growing cells. These observations suggest that the mismatch-repair deficiency of mutD strains results from a saturation of the mutHLS-mismatch-repair system by an excess of primary DNA replication errors due to the proofreading defect.     

Escherichia Coli Mutator Mutd5 Is Defective in the Muthls Pathway of DNA Mismatch Repair

We have previously reported that the Escherichia coli mutator strain mutD5 was defective in the correction of bacteriophage M13mp2 heteroduplex DNA containing a T.G mismatch. Here, this defect was further investigated with regard to its interaction with the mutHLS pathway of mismatch repair. A set of 15 different M13mp2 heteroduplexes was used to measure the mismatch-repair capability of wild-type, mutL and mutD5 cells. Throughout the series, the mutD5 strain proved as deficient in mismatch repair as the mutL strain, indicating that the repair defect is similar in the two strains in both extent and specificity. [One exception was noted in the case a T.G mispair that was subject to VSP (Very Short Patch) repair. VSP repair was abolished by mutL but not by mutD.] Variation in the dam-methylation state of the heteroduplex molecules clearly affected repair in the wild-type strain but had no effect on either the mutD or mutL strain. Finally, mutDmutL or mutDmutS double-mutator strains were no more deficient in mismatch repair as were the single mutator strains. The combined results strongly argue that the mismatch-repair deficiency of mutD5 cells resides in the mutH,L,S-dependent pathway of mismatch repair and that the high mutation rate of mutD strains derives in part from this defect.     

Mechanisms of spontaneous mutagenesis: an analysis of the spectrum of spontaneous mutation in the Escherichia coli lacI gene

We have obtained via DNA sequence analysis a spectrum of 174 spontaneous mutations occurring in the lac I gene of Escherichia coli. The spectrum comprised base substitution, frameshift, deletion, duplication and insertion mutations, of which the relative contributions to spontaneous mutation could be estimated. Two thirds of all lacI mutations occurred in the frameshift hotspot site. An analysis of the local DNA sequence suggested that the intensity of this hotspot may depend on structural features of the DNA that extend beyond those permitted by the repeated tetramer at this site. Deletions comprised the largest non-hotspot class (37%). They could be divided into two subclasses, depending on whether they included the lac operator sequence; the latter was found to be a preferred site for deletion endpoints. Most of the deletions internal to the lacI gene were associated with the presence of directly or invertedly repeated sequences capable of accounting for their endpoints. Base substitutions comprised 34% of the non-hotspot events. Unlike the base substitution spectrum obtained via nonsense mutations, G . C----A . T transitions do not predominate. A new base substitution hotspot was discovered at position +6 in the lac operator; its intensity may reflect specific features of the operator DNA. IS1 insertion mutations contributed 12% of the non-hotspot mutations and occurred dispersed throughout the gene in both orientations. Since the lacI gene is not A + T-rich, the contribution of IS1 insertion to spontaneous mutation in general might be underestimated. Single-base frameshift mutations were found only infrequently. In general, they did not occur in runs of a common base. Instead, their occurrence seemed based on the "perfection" of direct or inverted repeats in the local DNA sequence. Three (tandem) duplication events were recovered. No repeated sequences were found that might have determined their endpoints.     

Introduction, rescue and expression of plasmid genes in mammalian cells and Escherichia coli

A shuttle-vector system is described for the study of mutational specificity in mammalian cells. Using a plasmid (pGKTK) carrying the E. coli galactokinase gene (gk) and the herpes simplex virus thymidine kinase gene (tk), we demonstrate the introduction of a foreign gene into the chromosome of a mammalian cell (TK- mouse fibroblasts) and its efficient rescue back into E. coli. This system makes use of two genes, each of which can expressed in both E. coli and mammalian cells, thereby permitting one marker to be the mutational target and the other to maintain stable integration in the host. In addition, expression of both genes in bacteria makes it possible to deletion map mutants to facilitate their sequencing. In the case of a putative single-copy transformant (T8), about half of the rescued plasmids are identical in size and restriction pattern to the original plasmid. Each of these expressed the tk gene, indicating the fidelity of the rescue system.     

Complex Frameshift Mutations Mediated by Plasmid Pkm101: Mutational Mechanisms Deduced from 4-Aminobiphenyl-Induced Mutation Spectra in Salmonella

We used colony probe hybridization and polymerase chain reaction/DNA sequence analysis to determine the mutations in ~2,400 4-aminobiphenyl (4-AB) +S9-induced revertants of the -1 frameshift allele hisD3052 and of the base-substitution allele hisG46 of Salmonella typhimurium. Most of the mutations occurred at sites containing guanine, which is the primary base at which 4-AB forms DNA adducts. A hotspot mutation involving the deletion of a CG or GC within the sequence CGCGCGCG accounted for 100 and 99.9%, respectively, of the reversion events at the hisD3052 allele in the pKM101 plasmid-minus strains TA1978 (uvr(+)) and TA1538 (δuvrB). In strain TA98 (δuvrB, pKM101), which contained the SOS DNA repair system provided by the pKM101 plasmid, ~85% of the revertants also contained the hotspot deletion; the remaining ~15% contained one of two types of mutations: (1) complex frameshifts that can be described as a -2 or + 1 frameshift and an associated base substitution and (2) deletions of the CC or GG sequences that flank the hotspot site (CCGCGCGCGG). We propose a misincorporation/slippage model to account for these mutations in which (1) pKM101-mediated misincorporation and translesion synthesis occurs across a 4-AB-adducted guanine; (2) the instability of such a mispairing and/or the presence of the adduct leads to strand slippage in a run of repeated bases adjacent to the adducted guanine; and (3) continued DNA synthesis from the slipped intermediate produces a frameshift associated with a base substitution. This model readily accounts for the deletion of the CC or GG sequences flanking the hotspot site, indicating that these mutations are, in fact, complex mutations in disguise (i.e., cryptic complex frameshifts). The inferred base-substitution specificity associated with the complex frameshifts at the hisD3052 allele (primarily G·C -> T·A transversions) is consistent with the finding that 4-AB induced primarily G·C -> T·A transversions at the hisG46 base-substitution allele. The model also provides a framework for understanding the different relative mutagenic potencies of 4-AB at the two alleles in the various DNA repair backgrounds of Salmonella.     

Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex

Two regions of the genome, a 1-kbp portion of the zeste locus and a 1.1- kbp portion of the yolk protein 2 locus, were sequenced in six individuals from each of four species: Drosophila melanogaster, D. simulans, D. mauritiana, and D. sechellia. The species and strains were the same as those of a previous study of a 1.9-kbp region of the period locus. No evidence was found for recent balancing or directional selection or for the accumulation of selected differences between species. Yolk protein 2 has a high level of amino acid replacement variation and a low level of synonymous variation, while zeste has the opposite pattern. This contrast is consistent with information on gene function and patterns of codon bias. Polymorphism levels are consistent with a ranking of effective population sizes, from low to high, in the following order: D. sechellia, D. melanogaster, D.mauritiana, and D. simulans. The apparent species relationships are very similar to those suggested by the period locus study. In particular, D. simulans appears to be a large population that is still segregating variation that arose before the separation of D. mauritiana and D. sechellia. It is estimated that the separation of ancestral D. melanogaster from the other species occurred 2.5-3.4 Mya. The separations of D. sechellia and D. mauritiana from ancestral D. simulans appear to have occurred 0.58- 0.86 Mya, with D. mauritiana having diverged from ancestral D. simulans 0.1 Myr more recently than D. sechellia.      

Mutability of bacteriophage M13 by ultraviolet light: Role of pyrimidine dimers

Summary The role of pyrimidine dimers in mutagenesis by ultraviolet light was examined by measuring the UV-induced reversion of six different bacteriophage M13 amber mutants for which the neighboring DNA sequences are known. The mutational response at amber (TAG) codons preceded by a guanine or adenine (where no pyrimidine dimer can be formed) were compared with those preceded by thymine or cytosine (where dimer formation is possible). Equivalent levels of UV-induced mutagenesis were observed at both kinds of sites. This observation demonstrates that there is no requirement for a pyrimidine dimer directly at the site of UV-induced mutation in this single-stranded DNA phage. UV irradiation of the phage was also performed in the presence of Ag⁺ ions, which specifically sensitize the DNA to dimer formation. The two methods of irradiation, when compared at equal survival levels (and presumably equal dimer frequencies), produced equivalent frequencies of reversion of the amber phage. We believe these results indicate that while the presence of pyrimidine dimers may be a prerequisite for UV mutagenesis, the actual mutagenic event can occur at a site some distance removed from a dimer.