Translocation by Type I R-M enzymes

Most DNA-based molecular motors are ‘linear-tracking’ motors and use the repetitive nature of the repeating nucleotide base-pair to enable them to move along DNA.  The best example, and one of the most closely studied at the single-molecule level, is RNA polymerase (Harada et al., 2001).  This enzyme is responsible for synthesis of messenger RNA (the reading intermediate between DNA and protein) and uses the energy of this synthesis reaction to enable movement along the DNA, reading the bases as it moves and copying them into a single chain RNA molecule.  Many other motors follow this pattern, but usually have different functions and as a consequence interact differently with DNA (e.g. DNA helicases are responsible for unwinding the two strands of DNA, DNA polymerase synthesises a new strand of DNA, DNA repair enzymes are able to detect and remove damaged bases in the DNA).

Figure 1                   

Single molecule studies of RNA polymerase (and other polymerases) have involved immobilisation of the motor (enzyme) onto a surface; the enzyme will then bind the DNA, which carries a bead at one end (Schafer et al., 1991).   This stage is then followed by measurement of the forces exerted on the bead, required to stall movement along the DNA, by means of an optical trap (effectively a laser light that ‘holds’ the bead at its point of focus).

Figure 2                   

However, there is another type of DNA-based molecular motor that interacts with a specific site on the DNA and then moves the remaining DNA toward that site.  These motors belong to a large superfamily (SF-II) of helicase-like enzymes (Flaus and Owen-Hughes, 2001) and are particularly well illustrated by type I restriction-modification (R‑M) enzymes, but also include type III R-M enzymes, chromatin remodelling factors and a few chimeric enzymes.  Type I R‑M enzymes are distinguished from other restriction enzymes by the fact that binding to an unmethylated recognition site on the DNA, elicits DNA cleavage at a distantly located, non-specific site on the same DNA molecule.  ATP, which is required for DNA restriction, fuels translocation of the distal DNA toward the recognition site (Figure 1).  Cleavage occurs when translocation is blocked (Figure 2 and Janscak et al., 1999b), which can be due to a collision with another type I R‑M enzyme, or, due to a lack of DNA to translocate (e.g. on circular DNA - Szczelkun et al., 1996).  Rotation is also an inevitable outcome of this translocation, as the regular binding surfaces on the DNA are arrayed helically and the translocating motor follows the helical groove.

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