PMID:8444906
| Citation |
Wang, J and Grossman, L (1993) Mutations in the helix-turn-helix motif of the Escherichia coli UvrA protein eliminate its specificity for UV-damaged DNA. J. Biol. Chem. 268:5323-31 |
|---|---|
| Abstract |
The Escherichia coli UvrA protein possesses a stretch of amino acids, 494 to 513, that matches the consensus sequence of the helix-turn-helix motif of many sequence-specific DNA binding proteins. It also has two zinc finger motif regions and two ATP binding sites. To study the potential roles of both helix-turn-helix and zinc finger motifs in the functioning of UvrA protein, random mutations were created in these motif regions by degenerate oligonucleotide-directed mutagenesis. Using this method, 12 single substitution mutants (eight in the helix-turn-helix motif region, one in the N-terminal zinc finger region, and three in the C-terminal zinc finger region) were isolated that failed to confer UV resistance in the E. coli strain deleted of the uvrA gene. One "hyper" UV-resistant mutant, G275A, was identified that conferred significantly more UV resistance than the wild type in the MH1-delta A strain. To further investigate the mechanism of failure of these mutant UvrA proteins to support nucleotide excision repair, two mutant UvrA proteins, G502D and V508D, were selected for purification and characterization, since they carry mutations at the positions offered as the putative constellation for the helix-turn-helix motif. The binding affinity of these two mutants for nonirradiated plasmid DNA was unaffected by the mutations. Both mutant proteins exhibited substantial ATPase activity, and together with the UvrB protein, they were capable of generating positively supercoiled plasmid DNA from the relaxed form in the presence of ATP and bacterial topoisomerase I. However, both mutant proteins failed to respond to UV damage in the filter binding assay and were incapable of forming 2 x SSC-resistant nucleoprotein complexes with UvrB protein on UV-irradiated plasmid DNA. Taking these properties together, it appears that the mutations in the helix-turn-helix motif region impaired the UvrA protein's ability to recognize UV damage, while its other activities were largely unaffected. Interestingly, ERCC-3, a human DNA repair protein, also has a similar helix-turn-helix motif. Given the highly conserved nature of repair proteins in general, this observation raises the possibility that both procaryotes and eucaryotes might use similar mechanisms to recognize damaged sites in their genomes. |
| Links | |
| Keywords |
Adenosine Triphosphatases/chemistry; Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/metabolism; Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Base Sequence; DNA/metabolism; DNA/radiation effects; DNA Damage; DNA, Superhelical; DNA, Viral; DNA-Binding Proteins/chemistry; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Hydrolysis; Molecular Sequence Data; Mutation; Protein Conformation; Sequence Homology, Amino Acid; Structure-Activity Relationship; Substrate Specificity; Ultraviolet Rays; Zinc Fingers/genetics |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<protect><annotationlinks/></protect>
Mutant alleles described in the paper
<protect>
| Mutant | Residue Change | Codon Change | UV Sensitivity |
|---|---|---|---|
|
2-13 |
Ser269 to Pro |
TCG to CCG |
S |
|
3-15 |
Arg494 to Pro |
CGT to CCT |
S |
|
3-12 |
Ala498 to Glu |
GCG to CAG |
S |
|
3-5 |
Gly502 to Asp |
GGT to GAT |
S |
|
4-27 |
Leu505 to Asp |
CTG to CGG |
S |
|
4-13 |
Val506 to Asp |
GTT to GAT |
S |
|
4-42 |
Val508 to Asp |
GTT to GAT |
S |
|
4-7 |
Tyr510 to Phe |
TAC to TTC |
S |
|
4-4 |
Asp513 to His |
GAC to CAC |
S |
|
5-12 |
Cys740 to Phe |
TGC to TTC |
S |
|
5-7 |
Cys742 to Tyr |
AAA to ATA |
S |
|
5-7 |
Lys750 to Ile |
TGT to TAT |
|
|
6-14 |
His754 to Tyr |
CAC to TAC |
S |
|
6-12 |
Cys763 to Phe |
TGC to TTC |
S |
|
6-61 |
Cys763 to Ser |
TGC to TCC |
R |
| edit table |
</protect>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
See also
References
See Help:References for how to manage references in EcoliWiki.
