Single-stranded telomere-binding protein employs a dual rheostat for binding affinity and specificity that drives function
| Autor: | Deborah S. Wuttke, Leslie W. Glustrom, Kenneth Lyon, Victoria Lundblad, Margherita Paschini, Nicholas V. Parsonnet, Cynthia M. Reyes, Tasha B. Toro |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Alanine Telomere-binding protein Multidisciplinary biology Chemistry Mutant Saccharomyces cerevisiae t-RPA Biological Sciences single-stranded DNA binding biology.organism_classification Biochemistry Telomere Cell biology 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology Cdc13 Nucleic acid telomere maintenance Binding selectivity DNA DNA-binding specificity |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | Significance Proteins that bind nucleic acids are frequently categorized as being either specific or nonspecific, with interfaces to match that activity. In this study, we have found that a telomere-binding protein exhibits a degree of specificity for ssDNA that is finely tuned for its function, which includes specificity for G-rich sequences with some tolerance for substitution. Mutations of the protein that dramatically impact its affinity for single-stranded telomeric DNA are lethal, as expected; however, mutations that alter specificity also impact biological function. Unexpectedly, we found mutations that make the protein more specific are also deleterious, suggesting that specificity and nonspecificity in nucleic acid recognition may be achieved through more nuanced mechanisms than currently recognized. ssDNA, which is involved in numerous aspects of chromosome biology, is managed by a suite of proteins with tailored activities. The majority of these proteins bind ssDNA indiscriminately, exhibiting little apparent sequence preference. However, there are several notable exceptions, including the Saccharomyces cerevisiae Cdc13 protein, which is vital for yeast telomere maintenance. Cdc13 is one of the tightest known binders of ssDNA and is specific for G-rich telomeric sequences. To investigate how these two different biochemical features, affinity and specificity, contribute to function, we created an unbiased panel of alanine mutations across the Cdc13 DNA-binding interface, including several aromatic amino acids that play critical roles in binding activity. A subset of mutant proteins exhibited significant loss in affinity in vitro that, as expected, conferred a profound loss of viability in vivo. Unexpectedly, a second category of mutant proteins displayed an increase in specificity, manifested as an inability to accommodate changes in ssDNA sequence. Yeast strains with specificity-enhanced mutations displayed a gradient of viability in vivo that paralleled the loss in sequence tolerance in vitro, arguing that binding specificity can be fine-tuned to ensure optimal function. We propose that DNA binding by Cdc13 employs a highly cooperative interface whereby sequence diversity is accommodated through plastic binding modes. This suggests that sequence specificity is not a binary choice but rather is a continuum. Even in proteins that are thought to be specific nucleic acid binders, sequence tolerance through the utilization of multiple binding modes may be a broader phenomenon than previously appreciated. |
| Databáze: | OpenAIRE |
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