The Mutational Specificity of Two Escherichia Coli Dnae Antimutator Alleles as Determined from Laci Mutation Spectra

In a companion study we have described the isolation of a series of mutants of Escherichia coli that replicate their DNA with increased fidelity. These mutants carry a mutation in the dnaE gene, encoding the α (polymerase) subunit of DNA polymerase III holoenzyme, which is responsible for the faithful replication of the bacterial chromosome. The mutants were detected as suppressors of the high mutability of a mutL strain (defective in postreplicative mismatch correction), in which mutations may be considered to arise predominantly from errors of DNA replication. To investigate the specificity of these antimutator effects, we have analyzed spectra of forward mutations in the N-terminal part of the lacI gene (i(-d) mutations) for two of the mutL dnaE derivatives (dnaE911 and dnaE915), as well as the control mutL strain. DNA sequencing of over 600 mutants revealed that in the mutL background both antimutator alleles reduce specifically transition mutations (A·T -> G·C and G·C -> A·T). However, the two alleles behave differently in this respect. dnaE911 reduces A·T -> G·C more strongly than it does G·C -> A·T, whereas the reverse is true for dnaE915. Second, dnaE911 does not appear to affect either transversion or frameshift mutations, whereas dnaE915 displays a distinct mutator effect for both. This mutator effect of dnaE915 for frameshift mutations was confirmed by the frequency of reversion of the trpE9777 frameshift mutation. The discovery that dnaE antimutator alleles possess distinct specificities supports the notion that DNA polymerases discriminate against errors along multiple pathways and that these pathways can be influenced independently.     

Spontaneous Mutation in the Escherichia Coli Laci Gene

To gain more detailed insight into the nature and mechanisms of spontaneous mutations, we undertook a DNA sequence analysis of a large collection of spontaneous mutations in the N-terminal region of the Escherichia coli lacI gene. This region of circa 210 base pairs is the target for dominant lacI mutations (i-d) and is suitable for studies of mutational specificity since it contains a relatively high density of detectable mutable sites. Among 414 independent i-d mutants, 70.8% were base substitutions, 17.2% deletions, 7.7% additions and 4.3% single-base frameshifts. The base substitutions were both transitions (60%) and transversions (40%), the largest single group being G.C----A.T (47% of base substitutions). All four transversions were observed. Among the 71 deletions, a hotspot (37 mutants) was present: an 87-bp deletion presumably directed by an 8-bp repeated sequence at its endpoints. The remaining 34 deletions were distributed among 29 different mutations, either flanked (13/34) or not flanked (21/34) by repeated sequences. The 32 additions comprised 29 different events, with only two containing a direct repeat at the endpoints. The single-base frameshifts were the loss of a single base from either repeated (67%) or nonrepeated (33%) bases. A comparison with the spectrum obtained previously in strains defective in DNA mismatch correction (mutH, mutL, mutS strains) yielded information about the apparent efficiency of mismatch repair. The overall effect was 260-fold but varied substantially among different classes of mutations. An interesting asymmetry was uncovered for the two types of transitions, A.T----G.C and G.C----A.T being reduced by mismatch repair 1340- and 190-fold, respectively. Explanations for this asymmetry and its possible implications for the origins of spontaneous mutations are discussed.     

DNA Polymerase II of Escherichia Coli in the Bypass of Abasic Sites in Vivo

The function of DNA polymerase II of Escherichia coli is an old question. Any phenotypic character that Pol II may confer upon the cell has escaped detection since the polymerase was discovered 24 yr ago. Although it has been shown that Pol II enables DNA synthesis to proceed past abasic sites in vitro, no role is known for it in the bypass of those lesions in vivo. From a study of phage S13 single-stranded DNA, we now report SOS conditions under which Pol II is needed for DNA synthesis to proceed past abasic sites with 100% efficiency in vivo. Overproduction of the GroES(+)L(+) heat shock proteins, which are members of a ubiquitous family of molecular chaperones, eliminated this requirement for Pol II, which may explain why the role of Pol II in SOS repair had eluded discovery. Mutagenesis accompanied SOS bypass of abasic sites when the original occupant had been cytosine but not when it had been thymine; the quantitative difference is shown to imply that adenine was inserted opposite the abasic sites at least 99.7% of the time, which is an especially strict application of the A-rule. Most, but not all, spontaneous mutations from Rif(s) to Rif(r), whether in a recA(+) or a recA(Prt(c)) cell, require Pol II; while this suggests that cryptic abasic lesions are a likely source of spontaneous mutations, it also shows that such lesions cannot be the exclusive source.     

A Temperature Sensitive Suppressor Mutation of Escherichia Coli

Abstract

In an Escherichia coli K 12 strain, with a Pho ^- nonsense mutation and the suppressor gene Su-4, we have induced a mutant with a temperature dependent alkaline phosphatase negative phenotype. This phenotype can be ascribed to a mutation mapping in the Su-4 gene that makes the suppression temperature sensitive.     

DNA Sequence Effects on Single Base Deletions Arising during DNA Polymerization in Vitro by Escherichia Coli Klenow Fragment Polymerase

Most single base deletions detected after DNA polymerization in vitro directed by either Escherichia coli DNA polymerase I or its Klenow fragment are opposite Pu in the template. The most frequent study, were previously found to be associated with the consensus template context 5'-PyTPu-3'. In this study, the predictive power of the consensus sequence on single base deletion frequencies was directly tested by parallel comparison of mutations arising in four related DNAs differing by a single base. G, a deletion hotspot within the template context 5'-TTGA-3', was substituted by each of the 3 other bases. Previous studies had shown that deletions opposite the G were frequent but that deletions opposite its neighboring A were never detected. Based on the predictions of the consensus, the substitution of T for G should produce frequent deletions opposite the neighboring A due to its new 5'-TTTA-3' template context. This prediction was fulfilled; no deletions of this A were detected in the other templates. The consensus further predicted that deletions opposite template C would be lower than those opposite either A or G at the same site and this prediction was also fulfilled. The C substitution also produced a new hotspot for 1 bp deletions 14 bp away. The new hotspot depends on quasi-palindromic misalignment of the newly synthesized DNA strand during polymerization; accurate, but ectopically templated synthesis is responsible for this mutagenesis. Mutations templated by quasi-palindromic misalignments have previously been recognized when they produced complex sequence changes; here we show that this mechanism can produce frequent single base deletions. The unique stimulation of misalignment mutagenesis by the C substitution in the template is consistent with the singular ability of C at that site to contribute to extended complementary pairing during the DNA misalignment that precedes mutagenesis.     

Marker-Dependent Recombination in T4 Bacteriophage. IV. Recombinational Effects of Antimutator T4 DNA Polymerase

Recombinational effects of the antimutator allele tsL42 of gene 43 of phage T4, encoding DNA polymerase, were studied in crosses between rIIB mutants. Recombination under tsL42-restricted conditions differed from the normal one in several respects: (1) basic recombination was enhanced, especially within very short distances; (2) mismatch repair tracts were shortened, while the contribution of mismatch repair to recombination was not changed; (3) marker interference at very short distances was augmented. We infer that the T4 DNA polymerase is directly involved in mismatch repair, performing both excision of a nonmatched single strand (by its 3' -> 5' exonuclease) and filling the resulting gap. A pathway for the mismatch repair was substantiated; it includes sequential action of endo VII (gp49) -> 3'->5' exonuclease (gp43) -> DNA polymerase (gp43) -> DNA ligase (gp30). It is argued that the marker interference at very short distances may result from the same sequence of events during the final processing of recombinational intermediates.     

DNA Inversions between Short Inverted Repeats in Escherichia Coli

Using site-specific mutagenesis in vitro, we have constructed Escherichia coli strains that allow the detection of the inversion of an 800-bp segment in the lac region. The invertible segment is bounded by inverted repeats of either 12 or 23 bp. Inversions occurring at these inverted repeats will restore the Lac+ phenotype. Inversions can be detected at both short homologies at frequencies ranging from 0.5 x 10(-8) to 1 x 10(-7). These events, which have been verified by DNA sequence analysis, are reduced up to 1000-fold in strains deficient for either RecA, RecB or RecC. They are not reduced in strains deficient in the RecF, J pathway. These results show that the RecB,C,D system can mediate rearrangements at short sequence repeats, and probably plays a major role in cellular rearrangements.     

Opposing Roles of the Holliday Junction Processing Systems of Escherichia Coli in Recombination-Dependent Adaptive Mutation

Aspects of the molecular mechanism of ``adaptive' mutation are emerging from one experimental system: reversion of an Escherichia coli lac frameshift mutation carried on a conjugative plasmid. Homologous recombination is required and the mutations resemble polymerase errors. Reports implicating a role for conjugal transfer proteins suggested that the mutation mechanism is ordinary replication error occurring during transfer synthesis, followed by conjugation-like recombination, to capture the replicated fragment into an intact replicon. Whereas conjugational recombination uses either of two systems of Holliday junction resolution, we find that the adaptive lac reversions are inhibited by one resolution system and promoted by the other. Moreover, temporary absence of both resolution systems promotes mutation. These results imply that recombination intermediates themselves promote the mutations.     

Enhanced Deletion Formation by Aberrant DNA Replication in Escherichia Coli

Repeated genes and sequences are prone to genetic rearrangements including deletions. We have investigated deletion formation in Escherichia coli strains mutant for various replication functions. Deletion was selected between 787 base pair tandem repeats carried either on a ColE1-derived plasmid or on the E. coli chromosome. Only mutations in functions associated with DNA Polymerase III elevated deletion rates in our assays. Especially large increases were observed in strains mutant in dnaQ, the ε editing subunit of Pol III, and dnaB, the replication fork helicase. Mutations in several other functions also altered deletion formation: the α polymerase (dnaE), the γ clamp loader complex (holC, dnaX), and the β clamp (dnaN) subunits of Pol III and the primosomal proteins, dnaC and priA. Aberrant replication stimulated deletions through several pathways. Whereas the elevation in dnaB strains was mostly recA- and lexA-dependent, that in dnaQ strains was mostly recA- and lexA-independent. Deletion product analysis suggested that slipped mispairing, producing monomeric replicon products, may be preferentially increased in a dnaQ mutant and sister-strand exchange, producing dimeric replicon products, may be elevated in dnaE mutants. We conclude that aberrant Polymerase III replication can stimulate deletion events through several mechanisms of deletion and via both recA-dependent and independent pathways.     

The Effect of Divalent Cations and Core Polymerase in the Abortive Initiation by Escherichia Coli Dna-Dependent Rna Polymerase Using Phosphonate Dinucleotide Primers

The effect of divalent Mg²⁺ and Mn²⁺ cations on the elongation of ApU, UpA and their 3'-O- and 5'-O-phosphonylmethyl analogues by RNA polymerase holoenzyme to the corresponding trinucleo-tides on a poly(dA-dT) template was investigated. In contrast to Mg^z+ ions, Mn²⁺ ions enhance abortive trinucleotide synthesis. This effect is more pronounced with phosphonylmethyl analogues. The core enzyme cannot catalyze the elongation of either (2'-5') UpA or phosphonylmethyl analogues. The localization of the divalent cation activator, as well as the role of the σ subunit at the catalytic centre of the holoenzyme, is discussed.     

Double-Stranded Gap Repair of DNA by Gene Conversion in Escherichia Coli

We demonstrated repair of a double-stranded DNA gap through gene conversion by a homologous DNA sequence in Escherichia coli. We made a double-stranded gap in one of the two regions of homology in an inverted orientation on a plasmid DNA molecule and introduced it into an E. coli strain which has the RecE system of recombination (genotype; sbcA23 recB21 recC22). We detected repair products by genetic selection. The repair products were those expected by the double-strand-gap repair model. Gene conversion was frequently accompanied by crossing over of the flanking sequences as in eukaryotes. This double-strand gap repair mechanism can explain plasmid recombination in the absence of an artificial double-stranded break reported in a companion study by Yamamoto et al.     

Antimutator role of the DNA glycosylase mutY gene in Helicobacter pylori

Helicobacter pylori has a highly variable genome with ongoing diversification via inter- and intragenomic recombination and spontaneous mutation. DNA repair genes modulating mutation and recombination rates that influence diversification have not been well characterized for H. pylori. To examine the role of putative base excision repair ung and mutY glycosylase and xthA apurinic/apyrimidinic endonuclease genes in H. pylori, mutants of each were constructed in strain JP26 by allelic exchange. Spontaneous mutation frequencies of JP26 mutY mutants, assessed by rifampin resistance, were consistently higher (26-fold) than that of the wild type, whereas the ung and xthA mutants showed smaller increases. In trans complementation of the JP26 mutY mutant restored spontaneous mutation frequencies to wild-type levels. In cross-species studies, H. pylori mutY complemented an Escherichia coli mutY mutant and vice versa. In contrast, the ung and mutY mutants did not show higher frequencies of intergenomic recombination or greater sensitivity to UV-induced DNA damage than the wild type. The H. pylori mutY open reading frame contains an eight-adenine homonucleotide tract; we provide evidence that this is subject to slipped-strand mispairing, leading to frameshifts that eliminate gene function. Our findings indicate that H. pylori possesses phase-variable base excision repair, consistent with a tension between repair and mutation.