UmuDAb shares only 37% identity with LexA, and this similarity is restricted to the self-cleaving carboxy-terminus, not the DNA-binding N-terminal domain of LexA (Fig. 1). Because ADP1 possesses a mutated umuC gene (Hare et al., 2006), and the Acinetobacter species capable of DNA damage–induced mutagenesis possess both umuDC and umuDAb genes (Hare et al., 2012), the ability of UmuDAb to participate in SOS mutagenesis is unknown. The unexpected observation that a homolog of the error-prone polymerase accessory, UmuD, regulates

genes in response to DNA damage highlights the need to determine a mechanism that ties UmuDAb action to the DNA damage response. We hypothesize that check details UmuDAb responds to DNA damage with self-cleavage. Determining whether UmuDAb self-cleaves in response to DNA damage, and by what mechanism, will help elucidate the function of UmuDAb

in the Acinetobacter DNA damage response as regulator and/or polymerase accessory. The E. coli strains used, and their genotypes relevant to this study, were AB1157 (wild type), 315 (AB1157 ΔumuD772::kan), AB2463 (AB1157 recA13), and DH5α (recA1). Both recA− alleles, which are missense point mutations at G160D (recA1) or L51F (recA13), are defective for all activities except ssDNA binding (Lauder & Kowalczykowski, 1993). QIAGEN’s EasyXpress Protein Synthesis PCR process was used to second amplify umuDAb from plasmid TSA HDAC pJH1, which contains umuDAb in its native chromosomal context (Hare et al., 2006). The umuDAb PCR product was cloned into XbaI and BamHI restriction sites of the Qiagen EasyExpress pIX3.0 vector to form plasmid pIX2. pIX2AtoY, pIX2GtoE, pIX2StoA, and pIX2KtoA resulted from site-directed mutagenesis of the umuDAb codons for A83, G84, S119, or K156 in pIX2 with the Stratagene QuikChange II kit. These

mutations were confirmed by double-stranded DNA sequencing of the plasmids. Descriptions of these strains and plasmids are in Table 1. Dewitt & Adelberg (1962) Penny Beuning, Northeastern University Howard-Flanders & Theriot (1966) Leslie Gregg-Jolly, Grinnell College Total protein cellular lysates were prepared starting with overnight cultures grown shaking in 3 mL of LB broth with ampicillin at 37 °C. Cultures were diluted 1 : 10 in LB plus ampicillin and grown while shaking for an additional 3 h at 37 °C to enter early exponential phase. After 3 h, the culture was split into half, with 2 μg mL−1 MMC added to one culture. Alternately, for UV treatment, 400 μL of cell culture was washed and resuspended in phosphate-buffered saline, put in a 5.3-cm-diameter watch glass and exposed to 200 J m−2 UV-C light (or a mock treatment), using a Stratagene UV Stratalinker in the dark. These UV-exposed samples were pelleted and resuspended in media containing 100 μg mL−1 ampicillin.