lon incompatibility associated with mutations causing SOS induction: null uvrD alleles induce an SOS response in Escherichia coli

Abstract

The uvrD gene in Escherichia coli encodes a 720-amino-acid 3'-5' DNA helicase which, although nonessential for viability, is required for methyl-directed mismatch repair and nucleotide excision repair and furthermore is believed to participate in recombination and DNA replication. We have shown in this study that null mutations in uvrD are incompatible with lon, the incompatibility being a consequence of the chronic induction of SOS in uvrD strains and the resultant accumulation of the cell septation inhibitor SulA (which is a normal target for degradation by Lon protease). uvrD-lon incompatibility was suppressed by sulA, lexA3(Ind(-)), or recA (Def) mutations. Other mutations, such as priA, dam, polA, and dnaQ (mutD) mutations, which lead to persistent SOS induction, were also lon incompatible. SOS induction was not observed in uvrC and mutH (or mutS) mutants defective, respectively, in excision repair and mismatch repair. Nor was uvrD-mediated SOS induction abolished by mutations in genes that affect mismatch repair (mutH), excision repair (uvrC), or recombination (recB and recF). These data suggest that SOS induction in uvrD mutants is not a consequence of defects in these three pathways. We propose that the UvrD helicase participates in DNA replication to unwind secondary structures on the lagging strand immediately behind the progressing replication fork, and that it is the absence of this function which contributes to SOS induction in uvrD strains.

FIG. 1

Molecular characterization of lon-103 and lon-104 mutations. (a) The line at the bottom depicts the physical map of the wild-type lon locus (4, 19) for the enzymes BglII (B), EcoRI (E), KpnI (K), and PstI (P); the open circle and arrow beneath the line denote, respectively, the promoter and transcribed region of the lon gene. Horizontal line segments of the lower and upper inverted triangles represent, respectively, IS186 and Tn10dCm; the vertices denote sites of insertion of these two elements in the lon-103 mutant and the lon-104 suppressor-bearing strain, respectively. The figure is drawn to the scale marked; the distance between the PstI site at the left and the BglII site is 2.42 kb. The contiguous region of DNA (delimited by PstI sites) corresponding to the insert cloned in plasmid pHYD138 is shown by heavy line segments. The recognition site for the Tn10dCm-specific sequencing primer (see the text) is marked by the solid circle. (b) Nucleotide sequence determined with a Tn10dCm-specific sequencing primer using pHYD138 DNA as a template. The extents of Tn10dCm, IS186, and lon sequences are indicated by double-headed arrows above the sequence; dashed lines have been used where the delimited ends do not represent the natural ends of the sequence in question. The right end of IS186 is as specified in the work of Sengstag et al. (50), and the sequence corresponding to the 23-bp right inverted repeat is boxed. The −10 region of the lon promoter (4, 11) is underlined, and the base representing the transcription start site is italicized.