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Inference of relationships in the ‘twilight zone’ of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII
Thus far, identification of functionally important residues in Type II restriction endonucleases (REases) has been difficult using conventional methods. Even though known REase structures share a fold and marginally recognizable active site, the overall sequence similarities are statistically insign...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2005
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548357/ https://www.ncbi.nlm.nih.gov/pubmed/15684412 http://dx.doi.org/10.1093/nar/gki213 |
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author | Pawlak, Sebastian D. Radlinska, Monika Chmiel, Agnieszka A. Bujnicki, Janusz M. Skowronek, Krzysztof J. |
author_facet | Pawlak, Sebastian D. Radlinska, Monika Chmiel, Agnieszka A. Bujnicki, Janusz M. Skowronek, Krzysztof J. |
author_sort | Pawlak, Sebastian D. |
collection | PubMed |
description | Thus far, identification of functionally important residues in Type II restriction endonucleases (REases) has been difficult using conventional methods. Even though known REase structures share a fold and marginally recognizable active site, the overall sequence similarities are statistically insignificant, unless compared among proteins that recognize identical or very similar sequences. Bsp6I is a Type II REase, which recognizes the palindromic DNA sequence 5′GCNGC and cleaves between the cytosine and the unspecified nucleotide in both strands, generating a double-strand break with 5′-protruding single nucleotides. There are no solved structures of REases that recognize similar DNA targets or generate cleavage products with similar characteristics. In straightforward comparisons, the Bsp6I sequence shows no significant similarity to REases with known structures. However, using a fold-recognition approach, we have identified a remote relationship between Bsp6I and the structure of PvuII. Starting from the sequence–structure alignment between Bsp6I and PvuII, we constructed a homology model of Bsp6I and used it to predict functionally significant regions in Bsp6I. The homology model was supported by site-directed mutagenesis of residues predicted to be important for dimerization, DNA binding and catalysis. Completing the picture of sequence–structure–function relationships in protein superfamilies becomes an essential task in the age of structural genomics and our study may serve as a paradigm for future analyses of superfamilies comprising strongly diverged members with little or no sequence similarity. |
format | Text |
id | pubmed-548357 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2005 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-5483572005-02-10 Inference of relationships in the ‘twilight zone’ of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII Pawlak, Sebastian D. Radlinska, Monika Chmiel, Agnieszka A. Bujnicki, Janusz M. Skowronek, Krzysztof J. Nucleic Acids Res Article Thus far, identification of functionally important residues in Type II restriction endonucleases (REases) has been difficult using conventional methods. Even though known REase structures share a fold and marginally recognizable active site, the overall sequence similarities are statistically insignificant, unless compared among proteins that recognize identical or very similar sequences. Bsp6I is a Type II REase, which recognizes the palindromic DNA sequence 5′GCNGC and cleaves between the cytosine and the unspecified nucleotide in both strands, generating a double-strand break with 5′-protruding single nucleotides. There are no solved structures of REases that recognize similar DNA targets or generate cleavage products with similar characteristics. In straightforward comparisons, the Bsp6I sequence shows no significant similarity to REases with known structures. However, using a fold-recognition approach, we have identified a remote relationship between Bsp6I and the structure of PvuII. Starting from the sequence–structure alignment between Bsp6I and PvuII, we constructed a homology model of Bsp6I and used it to predict functionally significant regions in Bsp6I. The homology model was supported by site-directed mutagenesis of residues predicted to be important for dimerization, DNA binding and catalysis. Completing the picture of sequence–structure–function relationships in protein superfamilies becomes an essential task in the age of structural genomics and our study may serve as a paradigm for future analyses of superfamilies comprising strongly diverged members with little or no sequence similarity. Oxford University Press 2005 2005-01-31 /pmc/articles/PMC548357/ /pubmed/15684412 http://dx.doi.org/10.1093/nar/gki213 Text en © The Author 2005. Published by Oxford University Press. All rights reserved. |
spellingShingle | Article Pawlak, Sebastian D. Radlinska, Monika Chmiel, Agnieszka A. Bujnicki, Janusz M. Skowronek, Krzysztof J. Inference of relationships in the ‘twilight zone’ of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII |
title | Inference of relationships in the ‘twilight zone’ of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII |
title_full | Inference of relationships in the ‘twilight zone’ of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII |
title_fullStr | Inference of relationships in the ‘twilight zone’ of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII |
title_full_unstemmed | Inference of relationships in the ‘twilight zone’ of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII |
title_short | Inference of relationships in the ‘twilight zone’ of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII |
title_sort | inference of relationships in the ‘twilight zone’ of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases bsp6i and pvuii |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548357/ https://www.ncbi.nlm.nih.gov/pubmed/15684412 http://dx.doi.org/10.1093/nar/gki213 |
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