The Helicase-Like Domains of Type III Restriction Enzymes Trigger Long-Range Diffusion Along DNA

Abstract

Helicases are ubiquitous ATPases with widespread roles in genome metabolism. Here we report a new functionality for ATPases with helicase-like domains, namely that ATP hydrolysis can trigger ATP-independent long-range protein diffusion on DNA in one dimension (1D). Specifically, using single-molecule fluorescence microscopy we show that the Type III restriction enzyme EcoP15I uses its ATPase to switch into a distinct structural state that diffuses on DNA over long distances and long times. The switching occurs only upon binding to the target site and requires hydrolysis of ~30 ATPs. This study defines the mechanism for these enzymes and shows how ATPase activity is involved in DNA target site verification and 1D signaling, roles that are common in DNA metabolism, for example, in nucleotide excision and mismatch repair.Helicases were defined classically as ATPases that use directional translocation to unwind duplex nucleic acids. More recently however, many "pseudo-helicases" have been described that posses amino acid motifs characteristic of helicases yet fulfill their cellular functions without obligate DNA unwinding (1) and instead are often translocases that move directionally on single-(2) or double-stranded (3,4) nucleic acids. Pseudo-helicases include the ATP-dependent bacterial Type I and III restriction enzymes (REs) which use ATP hydrolysis to communicate between two distant restriction sites on the same DNA (5,6). Only if both sites are unmethylated is the DNA considered "foreign" and destroyed by the endonuclease. Type III REs form a heterooligomeric complex of two subunits: Mod (for target site methylation) and Res (for ATPase and endonuclease activities). Because they only hemimethylate their asymmetric recognition sequences, these enzymes must signal the relative site orientation during communication (7): cleavage occurs only if the sites are in an inverted-repeat orientation, arranged either "head-to-head" (HtH) (7) or "tail-to-tail" (TtT) (8). Type III REs hydrolyze only a few tens of ATP per dsDNA break (9,10), suggesting either passive three-dimensional looping between sites with limited translocation (11) or diffusion in 1D following an ATP-dependent switch (Fig. S1A) To distinguish between the different communication models, we developed a real-time single-molecule assay using magnetic tweezers combined with TIRF microscopy (12) that can localize fluorescently-labeled enzymes on stretched 26 kbp DNA molecules containing two centrally-located HtH EcoP15I sites with 6 kbp intersite distance (Fig. 1A). EcoP15I was labeled with quantum dots on the C-terminus of the Res subunit (QR-EcoP15I), without compromising the biochemical activity (Fig. S2). While DNA binding by lone quantum dots was not detected, addition of QR-EcoP15I and ATP resulted in enzyme attachment at one ( Fig. 1B and movie S1) or two ( Fig. S3) specific recognition sites. Enzymes always arrived instantaneously at the sites, with no evidence for long-lived non-specific binding en route (N = 61). Following a delay of, on av...

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