Functional evaluation of the C-terminal region of bacteriophage T4 Rad50
| Autor: | Yang Gao, Scott W. Nelson, Haley E. Streff |
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| Rok vydání: | 2020 |
| Předmět: |
Models
Molecular 0301 basic medicine DNA repair Allosteric regulation Mutant Biophysics Biochemistry Viral Proteins 03 medical and health sciences chemistry.chemical_compound Adenosine Triphosphate 0302 clinical medicine Escherichia coli Bacteriophage T4 Binding site Molecular Biology Adenosine Triphosphatases Nuclease Binding Sites biology Hydrolysis DNA Cell Biology DNA-Binding Proteins DNA binding site enzymes and coenzymes (carbohydrates) Exodeoxyribonucleases 030104 developmental biology chemistry 030220 oncology & carcinogenesis Rad50 biology.protein biological phenomena cell phenomena and immunity Protein Binding |
| Zdroj: | Biochemical and Biophysical Research Communications. 526:485-490 |
| ISSN: | 0006-291X |
| Popis: | Bacteriophage T4 encodes orthologs of the proteins Rad50 (gp46) and Mre11 (gp47), which form a heterotetrameric complex (MR) that participates in the processing of DNA ends for recombination-dependent DNA repair. Crystal and high-resolution cryo-EM structures of Rad50 have revealed DNA binding sites near the dimer interface of Rad50 opposite of Mre11, and near the base of the coiled-coils that extend out from the globular head domain. An analysis of T4-Rad50 using sequenced-based algorithms to identify DNA binding residues predicts that a conserved region of positively charged residues near the C-terminus, distal to the observed binding sites, interacts with DNA. Mutant proteins were generated to test this prediction and their enzymatic and DNA binding activities were evaluated. Consistent with the predictions, the Rad50 C-terminal mutants had reduced affinity for DNA as measured by Rad50 equilibrium DNA binding assays and an increased Km-DNA as determined in MR complex nuclease assays. Moreover, the allosteric activation of ATP hydrolysis activity due to DNA binding was substantially reduced, suggesting that these residues may be involved in the communication between the DNA and ATP binding sites. |
| Databáze: | OpenAIRE |
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