The Need for Speed: Run-On Oligomer Filament Formation Provides Maximum Speed with Maximum Sequestration of Activity

Abstract

Here, we investigate an unusual antiviral mechanism developed in the bacterium Streptomyces griseus. SgrAI is a type II restriction endonuclease that forms run-on oligomer filaments when activated and possesses both accelerated DNA cleavage activity and expanded DNA sequence specificity. Mutations disrupting the run-on oligomer filament eliminate the robust antiphage activity of wild-type SgrAI, and the observation that even relatively modest disruptions completely abolish this anti-viral activity shows that the greater speed imparted by the run-on oligomer filament mechanism is critical to its biological function. Simulations of DNA cleavage by SgrAI uncover the origins of the kinetic advantage of this newly described mechanism of enzyme regulation over more conventional mechanisms, as well as the origin of the sequestering effect responsible for the protection of the host genome against damaging DNA cleavage activity of activated SgrAI. IMPORTANCE This work is motivated by an interest in understanding the characteristics and advantages of a relatively newly discovered enzyme mechanism involving filament formation. SgrAI is an enzyme responsible for protecting against viral infections in its host bacterium and was one of the first such enzymes shown to utilize such a mechanism. In this work, filament formation by SgrAI is disrupted, and the effects on the speed of the purified enzyme as well as its function in cells are measured. It was found that even small disruptions, which weaken but do not destroy filament formation, eliminate the ability of SgrAI to protect cells from viral infection, its normal biological function. Simulations of enzyme activity were also performed and show how filament formation can greatly speed up an enzyme’s activation compared to that of other known mechanisms, as well as to better localize its action to molecules of interest, such as invading phage DNA.

Indirect Readout of DNA Controls Filamentation and Activation of a Sequence-Specific Endonuclease

“…2B ). In previous experiments, this property has been shown to specifically benefit the enzyme’s ability to quickly sequester phage DNA within the oligomeric form for rapid cleavage 24 . Within the filament, SgrAI makes several inter-DBD contacts between asymmetric units.…”

“…Beyond the well-known cytoskeletal NTPases, filament formation by enzymes is a newly appreciated phenomenon, with the advantages and evolutionary driving forces yet to be fully understood. Proposed purposes of filament formation vary among enzymes and include rapid enzyme activation, sequestration of activity, buffering of activity, and even in functioning as cytoskeletal structures 5,23,32–35 . In the case of SgrAI, an extensive kinetic study has recently been performed showing that association of SgrAI/DNA complexes into the ROO filament is the rate determining step under most conditions, and is overcome only through high enzyme and DNA concentrations (obtainable in vitro ).…”

“…In the case of SgrAI, an extensive kinetic study has recently been performed showing that association of SgrAI/DNA complexes into the ROO filament is the rate determining step under most conditions, and is overcome only through high enzyme and DNA concentrations (obtainable in vitro ). In a biological context, and owing to local concentration effects, filament formation is expected to occur when two cleavage sites reside on the same contiguous DNA 21–23 . This effect is significant and is responsible for sequestering SgrAI activity on phage DNA and away from the host genome 23 .…”

“…These studies show that assembly of DBDs into the ROO filament is rate limiting when recognition sites are on separate DNA molecules, but fast when on the same contiguous DNA. Kinetic simulations show that this rate limiting step underlies the enzyme’s ability to sequester potentially damaging secondary site cleavage events onto invading phage DNA and away from the host, leaving host sites largely untouched 23 . The study also shows that DNA cleavage is fast, followed by a relatively slower disassembly of DBDs from the ROO filament.…”

“…Although slower than DNA cleavage, the rate of disassembly is still sufficiently fast to prevent trapping of cleaved DNA within the filament. Furthermore, simulations also show that the very nature of filament formation imparts beneficial properties to the enzyme, both in terms of faster enzyme kinetics and its ability to sequester DNA cleavage onto phage and away from host, providing protection against infection and host DNA damage, respectively 23 . Importantly, mutations disrupting the ROO filament (even moderately) abolish all protection provided by SgrAI against phage infection, indicating that the rapid speed provided by ROO filament formation is critical to anti-phage activity 23 .…”