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 ...