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

Abstract

DNA cleavage by the Type III Restriction–Modification enzymes requires communication in 1D between two distant indirectly-repeated recognitions sites, yet results in non-specific dsDNA cleavage close to only one of the two sites. To test a recently proposed ATP-triggered DNA sliding model, we addressed why one site is selected over another during cleavage. We examined the relative cleavage of a pair of identical sites on DNA substrates with different distances to a free or protein blocked end, and on a DNA substrate using different relative concentrations of protein. Under these conditions a bias can be induced in the cleavage of one site over the other. Monte-Carlo simulations based on the sliding model reproduce the experimentally observed behaviour. This suggests that cleavage site selection simply reflects the dynamics of the preceding stochastic enzyme events that are consistent with bidirectional motion in 1D and DNA cleavage following head-on protein collision.

Dissociation from DNA of Type III Restriction–Modification enzymes during helicase-dependent motion and following endonuclease activity

“…As shown previously (24), an increase in enzyme concentration leads to an increase in the number of Type III enzymes on the DNA and a corresponding increase in cleavage (Figure 2C and Supplementary Figure S1). Since the signal saturates <100%, there must be some dissociation of these enzymes (otherwise at the highest concentration, 300 nM EcoPI, we would expect approximately 150 EcoPI enzymes per DNA which would give >99% cleavage at the EcoP15I site).…”

Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes

“…Although the data here simplifies the ATPase scheme of the Type III RM enzymes to a single catalytic burst of ATP hydrolysis, it still remains unclear why multiple ATPs are consumed to produce the conformation change in the complex. Although other groups have presented data to support linear stepwise translocation over longer distances ( 25 – 28 ), we have yet to find evidence for such movement during the initiation process or during long-range communication itself ( 3 , 5 , 9 , 20 ). Instead we have speculated that the reason why multiple ATPs are consumed is due to mechanochemical uncoupling ( 6 ), where the helicase releases ADP/phosphate before the conformational switch can complete and another round of ATP binding/hydrolysis must take place to maintain the stressed state.…”

“…Sliding was both fast (∼16 × 10 6 random single nucleotide steps per second) and long-lived (the lifetime before dissociation from internal sites—endo-dissociation—was ∼200 s). However, sliding of EcoP15I off the fluorescent oligoduplex via the DNA ends (exo-dissociation) would decrease the sliding lifetime to microseconds ( 3 , 5 , 9 , 10 ). Exo-dissociation was thus taken to be near instantaneous following release from the site.…”

CgII cleaves DNA using a mechanism distinct from other ATP-dependent restriction endonucleases

“…The original binding orientation of the enzyme is maintained during the sliding and thus cleavage shows site orientation preference ( 5 , 8 , 21 ). On plasmid or linear DNA with sites in inverted repeat (either head-to-head or tail-to-tail), dsDNA cleavage can be activated by head-on collision between a sliding enzyme and an enzyme bound to a site ( 5 , 20 , 22 ). The efficiency of cleavage of linear DNA relative to circular DNA is poor as the sliding enzymes can dissociate via the DNA ends of the former ( 5 ).…”

“…The efficiency of cleavage of linear DNA relative to circular DNA is poor as the sliding enzymes can dissociate via the DNA ends of the former ( 5 ). Additionally, the relative cleavage efficiency of tail-to-tail sites on linear DNA can be lower if the enzyme binds tightly to its site ( 22 ). For the single site DNA or two site head-to-tail (HtT) DNA, in either linear or circular form, DNA cleavage is not observed as all collisions are in the incorrect ‘rear-end’ orientation and the nuclease domains cannot engage ( 5 , 20 ).…”