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Abstract
T7 endonuclease I is a dimeric nuclease that is selective for four-way DNA junctions. Previous crystallographic studies have found that the N-terminal 16 amino acids are not visible, neither in the presence nor in the absence of DNA. We have now investigated the effect of deleting the N-terminus completely or partially. N-terminal deleted enzyme binds more tightly to DNA junctions but cleaves them more slowly. While deletion of the N-terminus does not measurably affect the global structure of the complex, the presence of the peptide is required to generate a local opening at the center of the DNA junction that is observed by 2-aminopurine fluorescence. Complete deletion of the peptide leads to a cleavage rate that is 3 orders of magnitude slower and an activation enthalpy that is 3-fold higher, suggesting that the most important interaction of the peptide is with the reaction transition state. Taken together, these data point to an important role of the N-terminus in generating a central opening of the junction that is required for the cleavage reaction to proceed properly. In the absence of this, we find that a cruciform junction is no longer subject to bilateral cleavage, but instead, just one strand is cleaved. Thus, the N-terminus is required for a productive resolution of the junction. (C) 2012 Elsevier Ltd. All rights reserved.
Original language | English |
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Pages (from-to) | 395-410 |
Number of pages | 16 |
Journal | Journal of Molecular Biology |
Volume | 425 |
Issue number | 2 |
DOIs | |
Publication status | Published - 23 Jan 2013 |
Keywords
- BACTERIOPHAGE T7
- ESCHERICHIA-COLI
- RESOLVING ENZYME CCE1
- STRUCTURAL RECOGNITION
- REPLICATION FORKS
- HOMOLOGOUS RECOMBINATION
- GENETIC-RECOMBINATION
- HOLLIDAY JUNCTION
- SULFOLOBUS-SOLFATARICUS
- SACCHAROMYCES-CEREVISIAE
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