Unequal fidelity of leading strand and lagging strand DNA replication on the Escherichia coli chromosome

Abstract

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 reflect 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.

Figure 1

(A) Insertion of the lac operon into the attL site of the E. coli chromosome in two orientations with regard to the chromosomal replication origin oriC. The orientation in which the lac operon is transcribed in the same direction as the movement of the replication fork through the target is designated the right (R) orientation, whereas the left (L) orientation indicates lac transcription in a direction opposite to the movement of the replication fork. The thick arrows at OriC represent the two forks initiated at this site. (B) A more detailed drawing of the R and L orientations for the case of the lacZ CC106 allele that reverts by A⋅T → G⋅C transition (AAG → GAG codon change), along with the potential T⋅G and A⋅C mispairs that cause this transition in either orientation. The dashed arrow indicates the direction of lac transcription.