Type III restriction enzymes cleave DNA by long-range interaction between sites in both head-to-head and tail-to-tail inverted repeat

Abstract

Cleavage of viral DNA by the bacterial Type III RestrictionModification enzymes requires the ATP-dependent long-range communication between a distant pair of DNA recognition sequences. The classical view is that Type III endonuclease activity is only activated by a pair of asymmetric sites in a specific head-to-head inverted repeat. Based on this assumption and due to the presence of helicase domains in Type III enzymes, various motor-driven DNA translocation models for communication have been suggested. Using both single-molecule and ensemble assays we demonstrate that Type III enzymes can also cleave DNA with sites in tail-to-tail repeat with high efficiency. The ability to distinguish both inverted repeat substrates from direct repeat substrates in a manner independent of DNA topology or accessory proteins can only be reconciled with an alternative sliding mode of communication.diffusion | helicase | motor | switch A lmost every genetic process requires protein complexes to interact simultaneously with specific DNA sites or structures that are located at-a-distance along the genome, including events in DNA replication, repair, recombination, and transcription. In many cases these so-called "long-range communications" are independent of the relative orientation of the interacting sites on the DNA (1). E.g., gene activation by remote enhancer elements is associated with random DNA looping between regulatory elements, i.e., chromatin loop formation (2). Such reactions can occur on DNA of any topology, and can even occur between sites on separate DNA molecules providing the local concentration is sufficiently elevated (1). In other cases however, a successful interaction only occurs when the sites are located on the same DNA in a specific relative orientation. The reaction can then be said to have "site-orientation selectivity," which can be achieved using both NTP-independent or NTP-dependent pathways:Site-specific recombinases (SSRs) have provided a mechanistic framework for NTP-independent communication (3-5). For example, the transposon-encoded resolvases have a strong preference to recombine sites in direct repeat on the same DNA (6). Site-orientation selectivity is important as uncontrolled rearrangements of DNA sequences may result in loss of function. For all SSRs studied to-date, the long-range communication occurs by the sites interacting via thermally driven three-dimensional diffusion, i.e., DNA looping (7). However, to achieve site-orientation selectivity, the geometry of the resulting site-site synapse is biased by an energetic "filter" that can be a preference for DNA substrates with a particular topology (e.g., 8) or the requirement for accessory DNA-binding factors (e.g., 9). For example, recombination by the resolvases requires the capture of three DNA nodes which are significantly favored by negative supercoiling (6,8,(10)(11)(12).For many processes that are NTP-dependent, site-orientation selectivity comes from directional one-dimensional motion along DNA. A classical example is transcription-...

Extending the Range for Force Calibration in Magnetic Tweezers

“…6). Our method is therefore particularly recommended for applications involving high forces, limiting molecule lengths, or low frame rates, such as high-resolution tracking of molecular motors (6,24,26), mechanical sequencing of DNA (34), or highly parallelized measurements on many molecules (48)(49)(50). In general, our calibration scheme does not exhibit any disadvantages compared to calibrations based on the short-pendulum geometry.…”

Translocation, switching and gating: potential roles for ATP in long-range communication on DNA by Type III restriction endonucleases

“…Translocation without loop formation has also been suggested [18]. However, other studies did not find evidence for either long-lived DNA loops or directional translocation [12,19,20]. Alternatively, it was suggested that ATP is used to catalyse a conformational switch from DNA recognition to one-dimensional DNA diffusion (also known as ‘DNA sliding’) [8,19].…”

DNA translocation by type III restriction enzymes: a comparison of current models of their operation derived from ensemble and single-molecule measurements

“…The absence of apparent looping in the TPM assay when applied to the type III R/M enzymes is also cited as support for the MT model (13,14), but it is apparent that the data are not of sufficient resolution, when compared with the data obtained for the Ecl18kI type II restriction enzyme, to rule out looping (32). It is important to note that DNA looping and one-dimensional diffusion along a DNA contour are both fast events occurring on the millisecond timescale, much faster than the time resolution of the assays currently employed, be they single molecule or ensemble assays (1,38,39).…”