Sep 9, 1981 - RALPH R. MEYER,'* DIANE C. REIN,1 AND JEFFREY GLASSBERG2. Department ofBiological Sciences, University ofCincinnati, Cincinnati, ...
Vol. 150, No. 1
JOURNAL OF BACTERIOLOGY, Apr. 1982, p. 433435 0021-9193/82/040433-03$02.00/0
The Product of the lexC Gene of Escherichia coli Is SingleStranded DNA-Binding Protein RALPH R. MEYER,'* DIANE C. REIN,1 AND JEFFREY GLASSBERG2 Department ofBiological Sciences, University of Cincinnati, Cincinnati, Ohio 45221,1 and The Rockefeller University, New York, New York 100212
Received 9 September 1981/Accepted 23 November 1981
Extracts from lexC113 cells could not support phage G4 DNA-dependent replication unless supplemented with single-stranded DNA-binding protein. Purified lexC113 binding protein supported synthesis in a reconstituted replication assay, using purified proteins at 30 but not at 42°C, indicating that the product of the lexC113 gene is an altered single-stranded DNA-binding protein.
The lexC113 mutation was first discovered in Escherichia coli B by Greenberg and colleagues as a radiation-sensitizing mutation, which they designated exrB (5). This mutation resulted in sensitivity to both UV and y irradiation and to several chemical agents, such as methylmethane sulfonate. This mutation differs from another related radiation-sensitive mutation, exrA (lexA of K-12 strains), in several respects, including filamentation in lon cells (5, 8) and, most notably, temperature-sensitive DNA synthesis in exrB cells (7). Johnson (8), in a detailed genetic analysis of this mutation, showed that it is clearly not a lexA allele, although it mapped near lexA. He proposed that it be renamed lexC. Both lexA and lexC mutants share similar properties: both fail to induce prophage (3, 8), their DNA is rapidly degraded after irradiation (2, 3, 8), and both lack UV-induced mutagenesis (6, 12). Based on these observations, Johnson proposed that lexC is an additional regulator of recAdependent SOS functions (8). During a study of temperature-sensitive DNA synthesis mutants (10), we identified one mutation which produced a temperature-sensitive single-stranded DNA-binding protein (SSB), which we designated ssb-1. Upon genetic analysis, the locus for ssb was mapped at 90.8 min in the same region as lexC (4). The ssb-l and lexC113 mutations produce similar phenotypes: both are temperature sensitive for DNA replication (7, 10, 11), UV sensitive (4, 13), defective in prophage induction (13), and fail to induce normal levels of recA protein after DNA damage (2; R. R. Meyer, D. W. Voegele, S. M. Ruben, D. C. Rein, and J. M. Trela, Mutat. Res., in press). We have previously suggested that ssb-l and lexC113 are allelic (4). In this note, we show that the two loci are identical since lexC113 cells produce a temperature-sensitive SSB. The E. coli B strain PAM5779 and its parent
strain WP2 were obtained from B. F. Johnson (Palo Alto Institute for Medical Research). The mutation was transduced into an E. coli K-12 background (C600), using a TnlO transposon and Pl transduction similar to the procedures detailed for the ssb-l mutation (4). These lexC strains were tested for binding protein deficiency by a phage G4 DNA complementation assay (10). Phage G4 DNA replication requires only three proteins: SSB, primase, and DNA polymerase III holoenzyme (15). Ammonium sulfate fractions prepared from extracts of wild-type strains WP2 or JGC247 contain these components and are able to support G4 DNA-dependent replication (Table 1). Extracts prepared from lexC113 cells, however, are inactive. Supplementation with either purified primase or DNA polymerase III holoenzyme had no effect, but addition of SSB restored DNA synthetic ability (Table 1). These results are identical to those obtained with ssb-1 extracts (4, 10) and clearly indicate that lexC strains are deficient in functional SSB. To confirm that the lexC113 allele specifies an altered binding protein, SSB was purified from PAM5779 cells by a procedure similar to that used to purify ssb-l binding protein (11). Phage G4 DNA replication (singlestranded DNA to replicative form) assays using normal or lexC113 SSB were carried out with purified proteins as shown in Table 2. Normal binding protein was effective in supporting replication at either 30 or 42°C, and the reaction was complete in 5 min. In contrast, with lexC SSB, the reaction at 42°C was only 5% of that at 30°C. Moreover, higher concentrations of mutant SSB were needed, and both the rate and extent of the reaction were diminished. These results are similar to those previously observed with binding protein prepared from ssb-l cells (10, 11). The data presented here establish that the lexC113 mutation codes for a single-stranded DNA-binding protein altered in its ability to
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NOTES
TABLE 1. Complementation by purified replication proteins of phage G4 DNA-dependent synthesis in extracts of E. coliP
xtrainusdto extract
S used for Strain
.. Addition Addltlon
TABLE 2. Temperature sensitivity of 1exC113 single-stranded DNA-binding protein in a reconstituted G4 DNA replication assay'
Nucleotides
incorporated (pmol)
WP2 (wild-type B) None 64 Primase 66 DNA polymerase III 64 holoenzyme SSB 59 PAM5779 (lexC113) None
