Genetic Interactions between the Escherichia coli umuDC Gene ...

More documents

Recommendations

Info

2904 SUTTON ET AL. J. BACTERIOL. FIG. 3. Relative position of each deduced amino acid substitution in the crystal. (A and B) Worm representation of the clamp showing the positions of missense mutations as viewed from the face bearing the extruding C-terminal tails of each protomer (A), and the side (B), corresponding to a 90° rotation of that shown in panel A. The crystal structure of the homodimeric clamp, solved by Kong et al. (21), is shown with one protomer in green and the other in blue. Positions of deduced amino acid substitutions are in red. The complex clamp loader and the subunit of Pol III are both believed to interact with the face of bearing the extruding C-terminal tails (B). See the text for further details. (C through G) Surface representations of the crystal in successive rotations of 45° each (i.e., 45°, 90°, 135°, and 180°, respectively) relative to the angle shown in panel A. This view bears the extruding C-terminal tails and is arbitrarily defined as 0°. Again, one protomer is in green and the other is in blue, and positions of deduced amino acid substitutions are in red. Note that not all of the missense mutations (for example, E204) are easily visible in the surface representation. This is because their position either is located just below the surface or is obstructed by other surface features of the clamp in the views shown. This figure was generated with GRASP (Molecular Simulations, Inc.) using the atom coordinates of the crystal (2POL) from the Protein Data Bank and a Silicon Graphics R10000 workstation. Downloaded from jb.asm.org at Harvard Libraries on February 1, 2009 dnaN alleles described in this report retains at least some biological activity with respect to DNA replication in vivo. However, their apparent inability to function in chromosomal replication at a level adequate for viability when expressed at the normal, physiological level suggests that it is difficult to genetically separate the replication function of from its ability to interact with the umuDC gene products. DISCUSSION Interactions of UmuD 2 C with components of Pol III help to enable a DNA damage checkpoint control. The cold sensitivity conferred by elevated levels of UmuD 2 C, a phenomenon that apparently is due to the inappropriate expression of their DNA damage checkpoint function (59), is exacerbated by overexpression of the processivity clamp (Table 2). This observation, taken together with our previous findings that overexpression of the ε proofreading subunit of Pol III or deletion of its structural gene (dnaQ) suppresses umuDC-mediated cold sensitivity whereas overexpression of each of the other eight Pol III subunits (, , , , , , , and ) does not (56), suggests that UmuD 2 C associates with the replisome via direct interactions with ε and and that these interactions serve to antagonize the DNA polymerase activity of Pol III, thereby arresting DNA synthesis (30, 37).
VOL. 183, 2001 NOVEL dnaN ALLELES DEFECTIVE IN INTERACTION WITH umuDC 2905 TABLE 5. Abilities of novel dnaN alleles to complement the temperature sensitivity of the dnaN59(Ts) E. coli strain HC123 Plasmid dnaN allele HC123 transformants/ml (42°C/30°C) a FIG. 4. Steady-state levels of mutant proteins in the absence of IPTG. The steady-state level of each mutant protein was measured in the absence of added IPTG by Western blot analysis using polyclonal antibodies specific to and chemiluminescent detection (Tropix) as described previously (36, 57). Approximately 10 8 cells of each strain grown in LB medium at 30°C to mid-exponential phase (optical density at 600 nm, 0.5) were electrophoresed in a sodium dodecyl sulfate–15% polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane as described previously (36, 57). Lane 1, AB1157(pJRC210); lane 2, AB1157(pBR322); lane 3, HC123(pJRC210); lane 4, HC123(pBR322); lane 5, HC123(pJRCHA- 4.1); lane 6, HC123(pJRCHA-5.1); lane 7, HC123(pJRCHA-8.1); lane 8, HC123(pJRCHA-5G11); lane 9, HC123(pJRCHA-8I11); lane 10, HC123(pJRCHA-7.1); lane 11, HC123(pJRCHA-6F11); and lane 12, HC123(pJRCHA-6.2). E. coli AB1157 and HC123 are described in Table 1; plasmids are described in Tables 1 and 3. Note that under the conditions used in this analysis, endogenous levels of expressed from the chromosomal dnaN allele (AB1157) (lane 2) were only barely detectable, while those expressed from the dnaN59(Ts) allele (HC123) (lane 4) were not detectable. Overexpression of also conferred a severe cold-sensitive growth phenotype upon a strain expressing moderately elevated levels of the umuDC gene products. Such a strain is not otherwise normally cold sensitive (59), although higher-level overexpression of UmuD 2 C does confer a cold sensitivity (38). We have previously suggested that the cold sensitivity conferred by higher-level overexpression of umuDC is a result of the ability of higher levels of UmuD 2 C to mimic the TABLE 4. Novel dnaN alleles deficient in conferring cold sensitivity upon a umuDC-expressing strain are unable to exacerbate the cold sensitivity of a umuDC-expressing strain Plasmid dnaN allele AB1157 transformants/ml (30°C/42°C) a with: pGY9738 (o c 1umuDC) pGY9739 (o c 1umuDC) pBR322 None 0.98 b 3.5 10 3b pJRC210 dnaN 2.6 10 4b 5.1 10 4b pJRCHA-4.1 Q61K 0.71 1.7 10 3 pJRCHA-5.1 S107L 0.37 1.5 10 3 pJRCHA-8.1 D150N 0.11 1.7 10 3 pJRCHA-5G11 G157S 0.83 0.25 pJRCHA-8I11 V170M 0.43 1.2 10 3 pJRCHA-7.1 E202K 0.47 2.7 10 2 pJRCHA-6F11 M204K 0.98 0.15 pJRCHA-6.2 P363S 0.30 3.5 10 3 a AB1157 bearing either pGY9738 or pGY9739 was transformed with either pBR322 or the indicated pJRC210 derivative. The ratio of CFU obtained at 30°C relative to those obtained at 42°C was measured as described in Table 2, footnote c. Transformation efficiencies at 42°C for all strains were on the order of 10 5 to 10 6 CFU per g of plasmid DNA. These results indicate that overexpression of either the umuD or umuDC gene products with or without simultaneous overexpression of is not lethal at 42°C under these conditions. b This value is the same as that reported in Table 2 and is included here for comparison to the eight novel dnaN alleles. pBR322 None 8.6 10 4 pJRC210 dnaN 1.11 pJRCHA-4.1 Q61K 0.71 b pJRCHA-5.1 S107L 1.02 pJRCHA-8.1 D150N 1.10 pJRCHA-5G11 G157S 0.86 b pJRCHA-8I11 V170M 0.95 pJRCHA-7.1 E202K 1.01 pJRCHA-6F11 M204K 0.92 pJRCHA-6.2 P363S 0.95 a The ratio of CFU obtained at 42°C to those obtained at 30°C was measured as described in Table 2, footnote c. Transformation efficiencies were on the order of 10 5 to 10 6 CFU per g of plasmid DNA. b This strain formed small colonies at 42°C, suggesting that the plasmid was slightly less efficient than pJRC210 at suppressing the temperature sensitivity of E. coli HC123. UmuD 2 C-dependent DNA damage checkpoint control (59). Thus, we now suggest that the -expressing plasmid confers cold sensitivity upon an E. coli strain expressing moderate levels of umuDC (a strain that is not normally cold sensitive [59]) by facilitating the ability of moderate levels of umuDC to mimic the DNA damage checkpoint function of UmuD 2 C, which presumably involves, at least in part, interaction of UmuD 2 C with (57, 59). We exploited the ability of the -expressing plasmid (pJRC210) to confer cold sensitivity upon a umuDC-expressing strain to identify eight unique and novel missense dnaN mutations. The fact that these plasmid-borne mutant dnaN alleles could complement the temperature sensitivity of a dnaN59(Ts) mutant (Table 5) suggests that when overexpressed, these mutant proteins retain a partial ability to participate in normal DNA replication. Some of the dnaN alleles described in this report (i.e., G157S, M204K, and, to a lesser extent, E202K) were able to suppress the inherent cold sensitivity conferred by elevated levels of the umuDC gene products in the absence of elevated levels of (Table 4), suggesting that their presence in the replisome confers upon it an immunity to the replication block normally imposed by elevated levels of UmuD 2 C. However, a definitive answer to the question of whether or not these mutant proteins are deficient in interaction with UmuD 2 C must await their biochemical characterization. The eight mutant proteins described in this report bear substitutions of amino acid residues located at or very close to the surface of the molecule (Fig. 3C to G), based on the crystal structure of the clamp reported by Kong et al. (21). Some of the native residues that were mutated are not easily visible in the surface representations of the clamp. This is because their position either is located just below the surface or is obstructed by other surface features of the clamp in the views shown. With respect to the fact that all of the mutations were located at or very close to the surface of the molecule, it is worthwhile emphasizing the fact that all of the mutations correspond to substitutions of a comparatively larger residue in place of the smaller, native residue (e.g., G157S). Thus, all of the mutant proteins described in this report are expected to have a slightly altered surface relative to that of the wild-type Downloaded from jb.asm.org at Harvard Libraries on February 1, 2009
Page 1 and 2: JOURNAL OF BACTERIOLOGY, May 2001,
Page 3 and 4: VOL. 183, 2001 NOVEL dnaN ALLELES D
Page 7: VOL. 183, 2001 NOVEL dnaN ALLELES D
Page 13: VOL. 183, 2001 NOVEL dnaN ALLELES D

Genetic Interactions between the Escherichia coli umuDC Gene ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?