PMID:18248777
| Citation |
Croteau, DL, DellaVecchia, MJ, Perera, L and Van Houten, B (2008) Cooperative damage recognition by UvrA and UvrB: identification of UvrA residues that mediate DNA binding.DNA Repair (Amst.) 7:392-404 |
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| Abstract |
Nucleotide excision repair (NER) is responsible for the recognition and removal of numerous structurally unrelated DNA lesions. In prokaryotes, the proteins UvrA, UvrB and UvrC orchestrate the recognition and excision of aberrant lesions from DNA. Despite the progress we have made in understanding the NER pathway, it remains unclear how the UvrA dimer interacts with DNA to facilitate DNA damage recognition. The purpose of this study was to define amino acid residues in UvrA that provide binding energy to DNA. Based on conservation among approximately 300 UvrA sequences and 3D-modeling, two positively charged residues, Lys680 and Arg691, were predicted to be important for DNA binding. Mutagenesis and biochemical analysis of Bacillus caldontenax UvrA variant proteins containing site directed mutations at these residues demonstrate that Lys680 and Arg691 make a significant contribution toward the DNA binding affinity of UvrA. Replacing these side chains with alanine or negatively charged residues decreased UvrA binding 3-37-fold. Survival studies indicated that these mutant proteins complemented a WP2 uvrA(-) strain of bacteria 10-100% of WT UvrA levels. Further analysis by DNase I footprinting of the double UvrA mutant revealed that the UvrA DNA binding defects caused a slower rate of transfer of DNA to UvrB. Consequently, the mutants initiated the oligonucleotide incision assay nearly as well as WT UvrA thus explaining the observed mild phenotype in the survival assay. Based on our findings we propose a model of how UvrA binds to DNA. |
| Links |
PubMed Online version:10.1016/j.dnarep.2007.11.013 |
| Keywords |
Adenosine Triphosphatases; Adenosine Triphosphate; DNA Damage; DNA Footprinting; DNA Helicases; DNA Repair; DNA, Bacterial; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Escherichia coli; Escherichia coli Proteins; Models, Molecular; Mutagenesis, Site-Directed; Protein Conformation |
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Main Points of the Paper
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Materials and Methods Used
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Phenotype Annotations
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| Phenotype of | Taxon Information | Genotype Information (if known) | Condition Information | OMP ID | OMP Term Name | ECO ID | ECO Term Name | Notes | Status |
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a mutation or genetic difference within a strain |
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Mutation caused a decrease in binding affinity to DNA, table 1. |
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a mutation or genetic difference within a strain |
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Mutation caused a decrease in binding affinity to DNA, table 1. |
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a mutation or genetic difference within a strain |
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Mutation caused a decrease in binding affinity to DNA, table 1. |
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a mutation or genetic difference within a strain |
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Double mutation caused a decrease in binding affinity to DNA more than single point mutations, table 1. |
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a mutation or genetic difference within a strain |
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Mutation caused a decrease in binding affinity to DNA, table 1. |
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Notes
References
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