Allosteric regulation of DNA cleavage and sequence-specificity through run-on oligomerization.
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.
National Resource for Automated Molecular Microscopy, Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Dmitry
D
Lyumkis
Heather
H
Talley
Andrew
A
Stewart
Santosh
S
Shah
Chad K
CK
Park
Florence
F
Tama
Clinton S
CS
Potter
Bridget
B
Carragher
Nancy C
NC
Horton
D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000648:ultrastructure; D001483:Base Sequence; D053837:DNA Cleavage; D059371:DNA, B-Form / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000648:ultrastructure; D004789:Enzyme Activation; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013305:Streptomyces griseus / Q000201:enzymology*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D059371:DNA, B-Form; C064435:endodeoxyribonuclease SgrAI; D015252:Deoxyribonucleases, Type II Site-Specific