Adaptive point mutation and amplification are induced responses to environmental stress, promoting genetic changes that can enhance survival. A specialized adaptive mutation mechanism has been documented in one Escherichia coli assay, but its enzymatic basis remained unclear. We report that the SOS-inducible, error-prone DNA polymerase (pol) IV, encoded by dinB, is required for adaptive point mutation in the E. coli lac operon. A nonpolar dinB mutation reduces adaptive mutation frequencies by 85% but does not affect adaptive amplification, growth-dependent mutation, or survival after oxidative or UV damage. We show that pol IV, together with the major replicase, pol III, can account for all adaptive point mutations at lac. The results identify a role for pol IV in inducible genetic change.

Upon starvation some Escherichia coli cells undergo a transient, genome-wide hypermutation (called adaptive mutation) that is recombination-dependent and appears to be a response to a stressful environment. Adaptive mutation may reflect an inducible mechanism that generates genetic variability in times of stress. Previously, however, the regulatory components and signal transduction pathways controlling adaptive mutation were unknown. Here we show that adaptive mutation is regulated by the SOS response, a complex, graded response to DNA damage that includes induction of gene products blocking cell division and promoting mutation, recombination, and DNA repair. We find that SOS-induced levels of proteins other than RecA are needed for adaptive mutation. We report a requirement of RecF for efficient adaptive mutation and provide evidence that the role of RecF in mutation is to allow SOS induction. We also report the discovery of an SOS-controlled inhibitor of adaptive mutation, PsiB. These results indicate that adaptive mutation is a tightly regulated response, controlled both positively and negatively by the SOS system.T he bacterial SOS response, studied extensively in Escherichia coli, is a global response to DNA damage in which the cell cycle is arrested and DNA repair and mutagenesis are induced (1). SOS is the prototypic cell cycle check-point control and DNA repair system, and because of this, a detailed picture of the signal transduction pathway that regulates this response is understood. A central part of the SOS response is the de-repression of more than 20 genes under the direct and indirect transcriptional control of the LexA repressor. The LexA regulon includes recombination and repair genes recA, recN, and ruvAB, nucleotide excision repair genes uvrAB and uvrD, the error-prone DNA polymerase (pol) genes dinB (encoding pol IV) (2) and umuDC (encoding pol V) (3), and DNA polymerase II (4, 5) in addition to many functions not yet understood. In the absence of a functional SOS response, cells are sensitive to DNA damaging agents.The signal transduction pathway leading to an SOS response (reviewed by ref. 6) ensues when RecA protein binds to singlestranded DNA (ssDNA), which can be created by processing of DNA damage, stalled replication, and perhaps by other means (7-9). The ssDNA acts as a signal that activates an otherwise dormant co-protease activity of RecA, which allows activated RecA (called RecA*) to facilitate the proteolytic self-cleavage of the LexA repressor, thus inducing the LexA regulon (10). Activated RecA also facilitates the cleavage of phage repressors used to maintain the quiescent, lysogenic state, and UmuD, creating UmuDЈ, the subunit of UmuDЈC (pol V) that allows activity in trans-lesion error-prone DNA synthesis (6).An intriguing feature of the SOS response is inducible mutation (11, 12). LexA-repressed pol V participates in most UV mutagenesis, by inserting bases across from pyrimidine dimers (3). Pol IV is required for an indirect mutation phenomenon in which undamaged phage DNA ...

Background: Inserting transgenes into bacterial chromosomes is generally quite involved, requiring a selection for cells carrying the insertion, usually for drug-resistance, or multiple cumbersome manipulations, or both. Several approaches use phage λ red recombination, which allows for the possibility of mutagenesis of the transgene during a PCR step.