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

Abstract

Much insight into the interactions of DNA and enzymes has been obtained using a number of single-molecule techniques. However, recent results generated using two of these techniques—atomic force microscopy (AFM) and magnetic tweezers (MT)—have produced apparently contradictory results when applied to the action of the ATP-dependent type III restriction endonucleases on DNA. The AFM images show extensive looping of the DNA brought about by the existence of multiple DNA binding sites on each enzyme and enzyme dimerisation. The MT experiments show no evidence for looping being a requirement for DNA cleavage, but instead support a diffusive sliding of the enzyme on the DNA until an enzyme–enzyme collision occurs, leading to cleavage. Not only do these two methods appear to disagree, but also the models derived from them have difficulty explaining some ensemble biochemical results on DNA cleavage. In this ‘Survey and Summary’, we describe several different models put forward for the action of type III restriction enzymes and their inadequacies. We also attempt to reconcile the different models and indicate areas for further experimentation to elucidate the mechanism of these enzymes.

DNA cleavage site selection by Type III restriction enzymes provides evidence for head-on protein collisions following 1D bidirectional motion

“…However, there is still a considerable debate in the field as to whether or not DNA loops play any role in the communication scheme (1,20). A series of AFM studies argued for extensive looping being induced by passive 3D diffusive loop capture and by active 1D translocation driven loop expansion (12,13).…”

“…This ‘DNA sliding’ model can account for the low ATP consumption during cleavage, the site orientation selectivity, the force-independence of cleavage and the inhibitory effect of open DNA ends (see below). Nonetheless, the actual communication model for Type III REs is still being debated (20).

Figure 1.Sliding model for long-range communication on DNA by Type III restriction enzymes.

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High-speed atomic force microscopy coming of age

“…A recent article discussed these and other experimental results to reconcile different models and provided suggestions for further experiments [125].…”

Host-Mimicking Strategies in DNA Methylation for Improved Bacterial Transformation

“…Type III RM systems operate with multi-subunit machinery comprising two R subunits and two M subunits (R 2 M 2 ) [33]. Subunit M contains recognition domain for binding to specific sites and also a methyltransferase domain.…”

“…This fact makes it difficult to transpose the translocation model that is proposed in type I RM systems to type III RM systems. Thus, some alternative models have now been proposed [33].…”