Following DNA binding and recognition that the substrate DNA is unmethylated the R-M enzyme undergoes a conformational change (ATP acting as an allosteric effector) that results in a switch to restriction activity. The enzyme remains tightly bound to the recognition site, but can also attach to adjacent DNA (in cis) (Szczelkun et al., 1996) prior to translocation. Translocation requires hydrolysis of ATP and the enzyme follows the DNA helix, which results in production of supercoils within the expanding loop (Janscak & Bickle, 2000). This possesses a major topological problem for the restriction endonuclease - there must be an (as yet) unknown mechanism by which the enzyme can "unwind" the supercoils as they form, to ensure topological barriers do not prevent further movement of the DNA (Szczelkun et al., 1996).
Recently, it has been shown that anything that blocks DNA translocation will produce DNA cleavage. As described earlier, this is usually the collision of two translocating enzymes (Figure 1). However, on cccDNA this barrier can be topological (Szczelkun et al., 1996) and this reflected in the increased rate of cleavage of single-site, circular DNA substrates. Finally, it has also been demonstrated that Holliday junctions also block translocation leading to DNA cleavage (Janscak et al., 1999).
The rate of translocation was determined using an oligonucleotide displacement assay (Firman & Szczelkun, 2000). The measurement were based around the use of a radio-labelled triple-helix forming oligonucleotide, which is displaced from a set of plasmids that also have a single recognition sequence for EcoR124I positioned at varying distances from the triple-helix binding site. A simple gel retardation assay allows the time for 50% displacement to be assayed. Modelling of a process involving a large number of steps during the translocation process predicts a lag period, which will increase as the distance between the sR124 recognition sequence and the triple-helix binding site increases. A plot of the observed lag period against time allows the rate of translocation to be determined. These experiments were able to show that translocation was indeed bi-directional, highly processive and the rate of translocation was 400bp sec-1 in both directions.
In addition, the R1-complex of the EcoR124I endonuclease was also found to displace the triple-helix forming oligonucleotide. In this case modelling showed that the translocation was unidirectional, and less processive than the wild-type enzyme.
Further studies using displacement of other DNA-bound ligand (including biotin-streptavidin linkages) have shown that the motor can re-set after translocation and it seems likely that this re-setting process is responsible for the large ATPase activity associated with DNA cleavage. The mechanism for re-setting has been shown to be due to dissociation of the HsdR (motor) subunit from the bound enzyme, leaving the MTase bound to the DNA (Seidel et al 2005). This result arose from various single molecule studies of the EcoR124I enzyme as a molecular motor.
To the right is an animation showing a model of unidirectional translocation occurs:
Last modified on
21 September 2011
© Dr Keith Firman
Author Dr Keith Firman.