Allosteric regulation of DNA cleavage and sequence-specificity through run-on oligomerization

Dmitry Lyumkis; Heather Talley; Andrew Stewart; Santosh Shah; Chad K. Park; Florence Tama; Clinton S. Potter; Bridget Carragher; Nancy C. Horton

doi:10.1016/j.str.2013.08.012

Allosteric regulation of DNA cleavage and sequence-specificity through run-on oligomerization

Dmitry Lyumkis, Heather Talley, Andrew Stewart, Santosh Shah, Chad K. Park, Florence Tama, Clinton S. Potter, Bridget Carragher, Nancy C. Horton

Chemistry and Biochemistry

Research output: Contribution to journal › Article › peer-review

16 Scopus citations

Abstract

SgrAI is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200- to 500-fold by effector DNA, with a concomitant expansion of its DNA sequence specificity. Using single-particle transmission electron microscopy to reconstruct distinct populations of SgrAI oligomers, we show that in the presence of allosteric, activating DNA, the enzyme forms regular, repeating helical structures characterized by the addition of DNA-binding dimeric SgrAI subunits in a run-on manner. We also present the structure of oligomeric SgrAI at 8.6 Å resolution, demonstrating the conformational state of SgrAI in its activated form. Activated and oligomeric SgrAI displays key protein-protein interactions near the helix axis between its N termini, as well as allosteric protein-DNA interactions that are required for enzymatic activation. The hybrid approach reveals an unusual mechanism of enzyme activation that explains SgrAI's oligomerization and allosteric behavior.

Original language	English (US)
Pages (from-to)	1848-1858
Number of pages	11
Journal	Structure
Volume	21
Issue number	10
DOIs	https://doi.org/10.1016/j.str.2013.08.012
State	Published - Oct 8 2013

ASJC Scopus subject areas

Structural Biology
Molecular Biology

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Allosteric regulation of DNA cleavage and sequence-specificity through run-on oligomerization. / Lyumkis, Dmitry; Talley, Heather; Stewart, Andrew; Shah, Santosh; Park, Chad K.; Tama, Florence; Potter, Clinton S.; Carragher, Bridget; Horton, Nancy C.

In: Structure, Vol. 21, No. 10, 08.10.2013, p. 1848-1858.

Research output: Contribution to journal › Article › peer-review

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abstract = "SgrAI is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200- to 500-fold by effector DNA, with a concomitant expansion of its DNA sequence specificity. Using single-particle transmission electron microscopy to reconstruct distinct populations of SgrAI oligomers, we show that in the presence of allosteric, activating DNA, the enzyme forms regular, repeating helical structures characterized by the addition of DNA-binding dimeric SgrAI subunits in a run-on manner. We also present the structure of oligomeric SgrAI at 8.6 {\AA} resolution, demonstrating the conformational state of SgrAI in its activated form. Activated and oligomeric SgrAI displays key protein-protein interactions near the helix axis between its N termini, as well as allosteric protein-DNA interactions that are required for enzymatic activation. The hybrid approach reveals an unusual mechanism of enzyme activation that explains SgrAI's oligomerization and allosteric behavior.",

author = "Dmitry Lyumkis and Heather Talley and Andrew Stewart and Santosh Shah and Park, {Chad K.} and Florence Tama and Potter, {Clinton S.} and Bridget Carragher and Horton, {Nancy C.}",

note = "Funding Information: We would like to thank New England Biolabs for the kind gift of SgrAI enzyme and Arne Moeller for help with figure graphics. This project was supported by grants from the National Science Foundation (NSF 0744732 to F.T. and N.C.H.) and the National Center for Research Resources (RR017573) and the National Institute of General Medical Sciences (GM103310) from the National Institutes of Health (to B.C. and C.S.P.). ",

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AU - Tama, Florence

AU - Potter, Clinton S.

AU - Carragher, Bridget

AU - Horton, Nancy C.

N1 - Funding Information: We would like to thank New England Biolabs for the kind gift of SgrAI enzyme and Arne Moeller for help with figure graphics. This project was supported by grants from the National Science Foundation (NSF 0744732 to F.T. and N.C.H.) and the National Center for Research Resources (RR017573) and the National Institute of General Medical Sciences (GM103310) from the National Institutes of Health (to B.C. and C.S.P.).

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N2 - SgrAI is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200- to 500-fold by effector DNA, with a concomitant expansion of its DNA sequence specificity. Using single-particle transmission electron microscopy to reconstruct distinct populations of SgrAI oligomers, we show that in the presence of allosteric, activating DNA, the enzyme forms regular, repeating helical structures characterized by the addition of DNA-binding dimeric SgrAI subunits in a run-on manner. We also present the structure of oligomeric SgrAI at 8.6 Å resolution, demonstrating the conformational state of SgrAI in its activated form. Activated and oligomeric SgrAI displays key protein-protein interactions near the helix axis between its N termini, as well as allosteric protein-DNA interactions that are required for enzymatic activation. The hybrid approach reveals an unusual mechanism of enzyme activation that explains SgrAI's oligomerization and allosteric behavior.

AB - SgrAI is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200- to 500-fold by effector DNA, with a concomitant expansion of its DNA sequence specificity. Using single-particle transmission electron microscopy to reconstruct distinct populations of SgrAI oligomers, we show that in the presence of allosteric, activating DNA, the enzyme forms regular, repeating helical structures characterized by the addition of DNA-binding dimeric SgrAI subunits in a run-on manner. We also present the structure of oligomeric SgrAI at 8.6 Å resolution, demonstrating the conformational state of SgrAI in its activated form. Activated and oligomeric SgrAI displays key protein-protein interactions near the helix axis between its N termini, as well as allosteric protein-DNA interactions that are required for enzymatic activation. The hybrid approach reveals an unusual mechanism of enzyme activation that explains SgrAI's oligomerization and allosteric behavior.

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Allosteric regulation of DNA cleavage and sequence-specificity through run-on oligomerization

Abstract

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