The role of filamentation in activation and DNA sequence specificity of the sequence-specific endonuclease SgrAI

Abstract

Filament formation by metabolic, biosynthetic, and other enzymes has recently come into focus as a mechanism to fine-tune enzyme activity in the cell. Filamentation is key to the function of SgrAI, a sequence-specific DNA endonuclease that has served as a model system to provide some of the deepest insights into the biophysical characteristics of filamentation and its functional consequences. Structure-function analyses reveal that, in the filamentous state, SgrAI stabilizes an activated enzyme conformation that leads to accelerated DNA cleavage activity and expanded DNA sequence specificity. The latter is thought to be mediated by sequence-specific DNA structure, protein–DNA interactions, and a disorder-to-order transition in the protein, which collectively affect the relative stabilities of the inactive, non-filamentous conformation and the active, filamentous conformation of SgrAI bound to DNA. Full global kinetic modeling of the DNA cleavage pathway reveals a slow, rate-limiting, second-order association rate constant for filament assembly, and simulations of in vivo activity predict that filamentation is superior to non-filamenting mechanisms in ensuring rapid activation and sequestration of SgrAI's DNA cleavage activity on phage DNA and away from the host chromosome. In vivo studies demonstrate the critical requirement for accelerated DNA cleavage by SgrAI in its biological role to safeguard the bacterial host. Collectively, these data have advanced our understanding of how filamentation can regulate enzyme structure and function, while the experimental strategies used for SgrAI can be applied to other enzymatic systems to identify novel functional roles for filamentation.

Two-Metal Ion Mechanism of DNA Cleavage by Activated, Filamentous SgrAI

“…In recent years, it has become clear that many important enzymes form filaments with altered properties such as increased or decreased enzymatic rates, cooperativity, or sensitivity to allosteric effectors (1,63–65). Altering substrate specificity is less common (66), but exemplified by SgrAI, which forms filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity (6). The studies described herein add to our growing understanding of the mechanisms by which filamentation activates the DNA cleavage properties of SgrAI.…”

“…Combined with previous structures of filamentous and non-filamentous SgrAI, a clearer picture of the activated DNA cleavage mechanism has emerged. Future studies will be aimed at understanding how filamentation also expands the DNA sequence specificity of SgrAI, which may derive from DNA structure and energetics, as well as disorder-to-order in segments of the SgrAI enzyme (6,34).…”

“…To date, over 30 enzymes from diverse biochemical pathways and from organisms representing all domains of life have been shown to form filamentous assemblies (1,2). A subset of these enzymes have been subjected to detailed studies to determine how filamentation affects enzyme activity (6,7). Many of the same enzymes known to form filaments in vitro also form large self-assemblies in cells that are visible by fluorescence microscopy, also known as cytoophidia (1,2,5,(8)(9)(10)(11)(12)(13).…”

“…One of the best studied enzymes that forms filamentous assemblies is the type II restriction endonuclease SgrAI. SgrAI cleaves an 8 base pair recognition sequence, CR|CCGGYG (R=A or G, Y=C or T, | denotes cleavage site) producing "sticky" ends consisting of a 4 base pair overhang 5'CCGG (6,17).…”

“…One of the best studied enzymes that forms filamentous assemblies is the type II restriction endonuclease SgrAI. SgrAI cleaves an 8 base pair recognition sequence, CR|CCGGYG (R=A or G, Y=C or T, | denotes cleavage site) producing “sticky” ends consisting of a 4 base pair overhang 5’CCGG (6,17). The recognition sequence possesses degeneracy at the second and seventh nucleotide, which lead to a total of 3 different sequences in double-stranded DNA that are known as primary recognition sequences or primary sites.…”