RESEARCH Areas

Contributions of signature DNA helicase motifs to translocation and DNA unwinding

Type I restriction enzymes bind to specific, bi-partite DNA sequences and cleave in cis, up to several thousand base pairs away. Cleavage at sites distant to the initial binding site is facilitated by a bi-directional translocation of dsDNA by two supposedly, diametrically opposed HsdR subunits. The HsdR subunits confer on the enzyme that ability to translocate dsDNA. Consistent, these subunits contain seven conserved "helicase" motifs. This has led to the proposal that translocation utilizes a helicase mechanism that includes DNA strand separation.
To more clearly understand the importance of these signature motifs in DNA helicase function, we selected the type I restriction enzyme EcoR124I (recognition sequence 5'-GAAnnnnnnPuTCG-3'). The domain organization of the HsdR subunits is reminiscent of the RecB subunit of RecBCD enzyme (a SF-I DNA helicase), which has helicase motifs, coupled to an endonuclease domain. Since RecBCD utilizes a quantum inchworm mechanism, it is conceivable that EcoR124I does so as well. However, the conserved motifs in HsdR are more similar at the primary sequence level to those in helicase SF-II than in SF-I, and if the analogy to RecG (SF-II) applies, then perhaps EcoR124I does not strand separate, but instead translocates on the outside of the DNA double helix.
Understanding the mechanism of translocation and whether DNA unwinding occurs, are important issues for EcoR124I. If we determine that this enzyme does not strand separate but only translocates dsDNA, this will provide a means to studying translocation and associated energetics, in the absence of DNA unwinding.

A schematic of the proposed organization of EcoR124I enzyme bound to its recognition sequence on dsDNA. R-subunits are shown in red. These subunits are sub-divided into 3 domains: N = nuclease, H = helicase and C = Carboxyl terminus. TRD = DNA target recognition domains of the S subunit. The arrows indicate DNA translocation with loops forming between the R and M2S complexes.