PubMed · 6312259
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M Gold, A Becker, W Parris. 1983. The bacteriophage lambda terminase enzyme.. https://doi.org/10.1016/0076-6879(83)00054-3
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Antimutators are genetic mutants that produce mutations at reduced rates compared to the wild type strain. They are interesting because they may provide insights into the mechanisms by which spontaneous mutations occur. We have investigated a reported antimutator strain of Escherichia coli termed mud for its possible mechanism. The mud strain exhibits a decrease in both spontaneous mutagenesis and mutability with alkylated agents and base analogs. These types of DNA lesions are known to be the substrates for the E. coli methyl-directed mismatch repair encoded by the mutHLSU system. We investigated whether the putative antimutator effect results from the increased expression or activity of the mutHLSU system. To directly measure the mismatch repair capacity of mud cells, we have transfected them with phage lambda heteroduplexes and scored the fraction of mixed (unrepaired) infective centers. This transfection system has been used routinely to assay mismatch repair capacity in E. coli and other organisms. No difference between mud and wild type cells is observed. From the results of the experiments we conclude that the reported antimutator effect of mud does not result from enhanced mismatch repair capacity. This conclusion is consistent with recently published evidence that the mud effect does not represent a real antimutator effect, but is an artifact due to impaired growth of mud cells under certain selective conditions.
Using molecular dynamics simulations in explicit solvent, we investigated the behavior of a 50-bp DNA sequence containing the 434 bacteriophage operators OR1 and OR2 separated by an 8-bp spacer. Two simulations of 1 ns each were carried out, with DNA alone and with DNA complexed to dimers of the R1-69 DNA binding domain of the phage 434 cI repressor protein at the OR1 and OR2 sites. Strong correlations among average structural parameters are observed between our simulations and available experimental data for the bound OR1/OR2 subsites. In the free state, some differences appear between the three relevant fragments (OR1, the spacer, and OR2). Unbound OR1 exhibits a large, shallow major groove into which the base atoms protrude and is also bent toward the major groove. This structure is maintained because structural fluctuations are weak. Unbound OR2 resembles canonical B-DNA although the structural parameters show greater fluctuations, essentially due to a malleable step (the innermost CpA/TpG), absent in OR1. Complexation with the proteins slightly alters the base positions but strongly modifies the sugar and backbone motions. The most crucial repressor effects are changes in the flexibility of the OR1/OR2 sites. Structural fluctuations are enhanced for OR1, conferring a favorable energetic contribution to the OR1 binding, whereas they are reduced for OR2. Therefore, both structural and dynamic properties of DNA suggest OR1 is the most attractive site for the repressor, which may explain the different binding association constants observed for the OR1 and OR2 sites. Finally, we also investigated the impact of the protein on the DNA backbone dynamics and find that direct or indirect interactions facilitate the DNA structural variations required for achieving complementarity with the protein.
Redesign of the bacteriophage 434 Cro repressor was accomplished by using an in vivo genetic screening system to identify new variants that specifically bound previously unrecognized DNA sequences. Site-directed, combinatorial mutagenesis of the 434 Cro helix-turn-helix (HTH) motif generated libraries of new variants which were screened for binding to new target sequences. Multiple mutations of 434 Cro that functionally converted wild-type (wt) 434 Cro DNA binding-sequence specificity to that of a lambda bacteriophage-specific repressor were identified. The libraries contained variations within the HTH sequence at only three positions. In vivo and in vitro analysis of several of the identified 434 Cro variants showed that the relatively few changes in the recognition helix of the HTH motif of 434 Cro resulted in specific and tight binding of the target DNA sequences. For the best 434 Cro variant identified, an apparent K(d) for lambda O(R)3 of 1 nM was observed. In competition experiments, this Cro variant was observed to be highly selective. We conclude that functional 434 Cro repressor variants with new DNA binding specificities can be generated from wt 434 Cro by mutating just the recognition helix. Important characteristics of the screening system responsible for the successful identifications are discussed. Application of the techniques presented here may allow the identification of DNA binding protein variants that functionally affect DNA regulatory sequences important in disease and industrial and biotechnological processes.