We have investigated the question whether during chromosomal DNA replication in Escherichia coli the two DNA strands may be replicated with differential accuracy. This possibility of differential replication fidelity arises from the distinct modes of replication in the two strands, one strand (the leading strand) being synthesized continuously, the other (the lagging strand) discontinuously in the form of short Okazaki fragments. We have constructed a series of lacZ strains in which the lac operon is inserted into the bacterial chromosome in the two possible orientations with regard to the chromosomal replication origin oriC. Measurement of lac reversion frequencies for the two orientations, under conditions in which mutations ref lect replication errors, revealed distinct differences in mutability between the two orientations. As gene inversion causes a switching of leading and lagging strands, these findings indicate that leading and lagging strand replication have differential fidelity. Analysis of the possible mispairs underlying each specific base pair substitution suggests that the lagging strand replication on the E. coli chromosome may be more accurate than leading strand replication.The question as to how organisms duplicate their DNA with high accuracy is of fundamental interest. Previous studies have revealed the functioning of at least three separate steps, base selection, proofreading, and DNA mismatch repair, which, by their sequential action, are responsible for the low error rate of Ϸ10 Ϫ10 per base replicated (1, 2). The most detailed information about this process is available for the bacterium E. coli based on both enzymological and genetical data. Replication of the E. coli chromosome is performed by DNA polymerase III holoenzyme, an asymmetric dimeric enzyme composed of 18 subunits (10 distinct) that simultaneously replicates the leading and lagging strand of the replication fork (for review, see ref.3). It contains two polymerase core units, one for each strand, each consisting of three tightly associated subunits, ␣, , and . Of these, ␣ is the polymerase (dnaE gene product), (dnaQ gene product) is a 3Ј 3 5Ј exonuclease that performs an editing function, and is a small subunit of unknown function. Additional components of the holoenzyme include the subunit ( 2 ) that dimerizes the two cores, the ␤ subunit (␤ 2 ) that encircles the DNA and tethers each DNA polymerase to the DNA to ensure high processivity, and the five-subunit ␥ complex (␥, ␦, ␦Ј, , and ) that loads the ␤ rings onto the DNA.With regard to the fidelity of polymerase III holoenzyme, as studied both in vivo and in vitro, the main focus has been on the role of the ␣ and subunits. The ␣ (polymerase) subunit plays a critical role through the process of base selection, selecting with great preference correct nucleotides at the nucleotide insertion step. The subunit, in conjunction with the polymerase, is responsible for the subsequent proofreading step, in which by virtue of its 3Ј exonuclease activity incorrectly inserted ...

We have determined the DNA sequence changes in 487 spontaneous mutations in the N-terminal part of the lacI gene in mutH, mutL, and mutS strains of Escherichia coli. These strains display elevated spontaneous mutation rates because of a deficiency in the process of postreplicative mismatch correction. As a consequence the mutational spectra reveal the nature of spontaneous DNA replication errors. The spectra consist of base substitutions (75%) and single-base deletions (25%). Among the base substitutions, transitions (both A.T-*G.C and G'C->AT) are strongly favored over transversions (96% versus 4%). Large site-to-site differences are observed among identical base substitutions, presumably reflecting the modulating effects of neighboring bases. The single-base-deletion spectrum is dominated by a large hotspot at a run of adjacent identical base pairs, implying a Streisingerslippage mechanism. The data, when compared to a previously determined wild-type spectrum, also provide information on the specificity of the mismatch repair system. Escherichia coli strains that carry a mutation in the mutH, mutL, or mutS genes display elevated spontaneous mutation rates because they are defective in methylation-instructed DNA mismatch correction (1-8). This system is thought to follow the replication fork, scrutinizing the newly replicated DNA for mismatches resulting from errors of DNA replication. The mismatches are then corrected using the undermethylation (at GATC sites) of the newly synthesized strand to distinguish the correct from the incorrect half of the mismatch.Here, we have made use of the mismatch-repair-deficient strains to further investigate the mechanisms of mutation in E. coli. We previously reported in detail on the DNA sequence changes in a large collection of spontaneous mutants in the lacd gene in a wild-type strain (9). Diverse mutational classes were seen, presumably resulting from different mutational mechanisms. We now report on the DNA sequence changes in 487 mutants in mutH, mutL, and mutS strains. Because errors of DNA replication in these strains are no longer corrected, the data provide us with an intimate view on the nature of in vivo DNA replication errors. The data, in conjunction with the wild-type data, also allow an estimation of the efficiency of the mismatch-repair system for several mutational classes. MATERIALS AND METHODSBacterial Strains. Escherichia coli strains NR3835 (ara, thi, trpE9777, Aprolac, F'prolac), NR3939 (ara, thi, mutH101, Aprolac, F'prolac), NR3940 (ara, thi, trpE9777, mutL101, Aprolac, F'prolac), and NR3996 (ara, thi, trpE9777, mutS-101, Aprolac, F'prolac), all derivatives of strain GM1 (10), were obtained from B. W. Glickman (York University, Toronto). The isolation and characterization of the mutator alleles has been described (2). The F'prolac carries the IQ(IacI) and L8(lacZ) promoter mutations. Strains CSH51, CSH52, and S90C have been described (10, 11).Media. Luria broth (LB) and minimal media were used as described (9). P-gal plates, used for the selec...

The mutagenic potential of apurinic sites in vivo has been studied by transfection of depurinated 4X174 DNA containing amber mutations into SOS-induced Escherichia coli spheroplasts. Mutagenicity is abolished by treatment of the depurinated DNA with an apurinic endonuclease from Hela cells, establishing the apurinic site as the mutagenic lesion. The frequency of copying apurinic sites in vitro was analyzed by measuring the extent of DNA synthesis using E. coli DNA polymerase I and avian myeloblastosis DNA polymerase. The inhibitionofDNA synthesis by apurinie sites was less with avian myeloblastosis DNA polymerase, suggesting that this error-prone enzyme copies apurinic sites with greater frequency. Consistent with this conclusion is the observation that, upon transfection into (normal) spheroplasts, the reversion frequency of depurinated 4X174 am3 DNA copied with avian myeloblastosis virus DNA polymerase is, much greater than that of the same DNA copied with E. coli DNA polymerase I. Sequence analysis of the DNA of 33 revertant phage produced by depurination indicates a preference for incorporation of deoxyadenosine opposite putative apurinic sites. The combined results indicate that mutagenesis resulting from apurinic sites is associated with bypass ofthese noncoding lesions during DNA synthesis.Depurination is the loss of purine bases from DNA through hydrolysis ofthe N-glycosylic bond that connects the base to the sugar-phosphate backbone. This process occurs spontaneously at significant rates. It has been estimated that 10,000 purines may be lost from the genome of a mammalian cell per 24-hr period (1). The presence of large amounts of apurinic endonuclease activity (2) and, possibly, insertase activity (3) in cells testifies to the potentially harmful effects of the loss of hereditary information through depurination. Apurinic sites also result from exposure of cells to various chemical carcinogens. Modification of bases, especially at positions N-3 and N-7 of purines (4) or position 0-2 of pyrimidines (5) We have studied the mutagenic potential of apurinic sites in various systems (8-10). Prokaryotic and eukaryotic DNA polymerases show increased misincorporation when copying synthetic polynucleotide templates containing apurinic sites (8). Depurination of 4X174 an3 DNA leads to enhanced mutagenesis when this DNA is copied in vitro by Escherichia coli DNA polymerase 1 (9). Finally, transfection ofdepurinated am3 DNA into E. coli spheroplasts is highly mutagenic for the phage when the spheroplasts are prepared from bacteria previously exposed to UV light (10). Presumably, UV irradiation induces an SOS response in the bacteria (II) which persists in the spheroplasts.Because mutagenesis in. SOS-induced cells is thought to be associated with an error-prone process that permits bypass of otherwise blocking lesions, we have studied in detail the relationship between the ability of an enzyme to polymerize past apurinic sites and the mutation frequency of its product DNA. This was done by comparing these ...