Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 425, Issue 2, Pages 395-410Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2012.11.029
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Funding
- Cancer Research UK
- BBSRC [BB/E001777/1] Funding Source: UKRI
- EPSRC [EP/J017094/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/E001777/1] Funding Source: researchfish
- Cancer Research UK [11722] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [EP/J017094/1] Funding Source: researchfish
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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.
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