SOS mutator activity: unequal mutagenesis on leading and lagging strands

Abstract

A major pathway of mutagenesis in Escherichia coli is mediated by the inducible SOS response. Current models of SOS mutagenesis invoke the interaction of RecA and UmuD'(2)C proteins with a stalled DNA replication complex at sites of DNA lesions or poorly extendable terminal mismatches, resulting in an (error-prone) continuation of DNA synthesis. The precise mechanisms of SOS-mediated lesion bypass or mismatch extension are not known. Here, we have studied mutagenesis on the E. coli chromosome in recA730 strains. In recA730 strains, the SOS system is expressed constitutively, resulting in a spontaneous mutator effect (SOS mutator) because of reduced replication fidelity. We investigated whether during SOS mutator activity replication fidelity might be altered differentially in the leading and lagging strand of replication. Pairs of recA730 strains were constructed differing in the orientation of the lac operon relative to the origin of replication. The strains were also mismatch-repair defective (mutL) to facilitate scoring of replication errors. Within each pair, a given lac sequence is replicated by the leading-strand machinery in one orientation and by the lagging-strand machinery in the other orientation. Measurements of defined lac mutant frequencies in such pairs revealed large differences between the two orientations. Furthermore, in all cases, the frequency bias was the opposite of that seen in normal cells. We suggest that, for the lacZ target used in this study, SOS mutator activity operates with very different efficiency in the two strands. Specifically, the lagging strand of replication appears most susceptible to the SOS mutator effect.

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 as 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) Presented is a more detailed drawing of the replication fork advancing (Left to Right) through the lacZ target of the CC105 allele that reverts by A⋅T → T⋅A transversion (GTG → GAG codon change), along with the potential A⋅A and T⋅T mispairs that can cause this transversion in L or R orientations. The dashed arrow indicates the direction of lac transcription. The assignment of A⋅A and T⋅T mispairs to either leading or lagging strand replication can be deduced as follows. The sequence 5′-AAT-GTG-AGT-3′ (underline, base to be mutated) represents the (+) strand lacZ coding sequence for this allele (32). The 5′ → 3′ direction of this sequence is, by necessity, also the direction of transcription. As defined above, in the R orientation (B, lower diagram) the direction of transcription has the same direction as the advancing replication fork. As a consequence, the 5′-AAT-GTG-AGT-3′ sequence is copied by the lagging-strand replication machinery. This places the T⋅T mispair in the lagging strand and the A⋅A mispair in the leading strand. In the L orientation (B, upper diagram) the situation is reversed. Table 4 of reference 27 delineates the corresponding mispairs for the other lacZ alleles used. Thus, for the R orientation, the CC104 (G⋅C→T⋅A) allele is characterized by (C⋅T)_lagging and (G⋅A)_leading, whereas the CC106 (A⋅T→G⋅C) allele is characterized by (A⋅C)_lagging and (T⋅G) _leading (for each mispair, the template base is stated first).