Involvement of outside DNA sequences in the major kinetic path by which EcoRI endonuclease locates and leaves its recognition sequence.

Abstract

We have examined the kinetics of the interaction between endodeoxyribonuclease EcoRI (EC 3.1.23.13) and nine linear DNA fragments that range in size between 34 and 6,200 base pairs and contain the EcoRI site of plasmid pBR322 in a central location. The kinetic parameters governing both formation and decay of specific endonucleaseDNA complexes increase 8-fold with increasing chain length over this size range. In contrast, equilibrium competition experiments demonstrated that the intrinsic affinity of endonuclease for its recognition sequence is independent of DNA chain length over this range. Thus, DNA sequences outside the recognition site enhance the rate at which EcoRI endonuclease locates or leaves its recognition site without affecting the intrinsic thermodynamic parameters of site-specific interaction. These results are consistent with a facilitated diffusion mechanism for specific DNA site location by this enzyme.DNA recognition sequences ofsite-specific proteins are embedded in a large background of nonspecific DNA. Nevertheless, such proteins locate their recognition sites by kinetically efficient processes, with reported apparent second-order association rate constants approaching or exceeding those expected for diffusion-limited collision of macromolecules (1). These surprising findings led to the suggestion that the effective target size for specific DNA-protein interaction might be much larger than the size of the recognition sequence (1, 2). In such models the protein is envisioned to interact with both specific and nonspecific DNA sequences, with interactions of the latter type being on the major kinetic path by which the protein locates its recognition site. Thus, random collisions between protein and DNA would initially favor formation of nonspecific complexes, which could then be converted to site-specific complexes by a facilitated diffusion mechanism in which the protein might, for example, "slide" along the polynucleotide via a random walk until the recognition sequence is encountered.Although theoretical treatments (2-4) have indicated that such a mechanism can account for the kinetic efficiency of sitespecific proteins, direct experimental support has been limited (1,(5)(6)(7)(8). A minimal test ofthis type ofmechanism requires proof of interaction of the protein in question with nonspecific sequences and demonstration that such interactions are involved in the path by which the protein locates and leaves its recognition site. In this paper we demonstrate that DNA sequences external to the recognition site markedly enhance the rate at which endodeoxyribonuclease EcoRI (EC 3.1.23.13) locates and leaves its recognition site. The external sequences, however, are without effect on the intrinsic equilibrium constant governing specific interactions. These findings, are in accord with a facilitated diffusion mechanism for specific DNA site location by this enzyme. A preliminary account of this work has been presented (5).MATERIALS AND METHODS Enzymes. EcoRI endonuclease was the homogeneou...

Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA

“…However, the actual path length for sliding, and the effect of salt on this process, are likely to vary from one protein to the next. Some DNA-binding proteins may stay in contact with the DNA for translocations over longer distances than BbvCI, as reported for certain proteins in low salt buffers (10,18), whereas others may dissociate after shorter distances. The key factor in determining the mean path length for 1D diffusion by a protein on DNA is its affinity for nonspecific DNA (8)(9)(10)(11).…”

“…However, to distinguish the schemes, it is necessary to vary not only the length of the DNA but also the affinity of the protein for nonspecific DNA (8,11), the latter normally being implemented by varying the salt concentration (20). Otherwise, the length dependency reveals that the nonspecific DNA is on the path to the specific site, but not the actual pathway (18). Observations of single protein molecules on DNA, by fluorescence or microscopy (21-23), have likewise been reconciled to sliding, although the distinction between the schemes requires a time resolution of Ͻ1 ms [because the process may cover 10 3 bp͞s (9)] and a spatial resolution of Ͻ1 nm (to see if the protein maintains contact as it takes 1-bp steps on the DNA).…”

“…However, much of the experimental data that support sliding can be reconciled to other schemes. For example, the effects of DNA length on the kinetics and͞or equilibria of the protein binding to its site are often correlated to 1D diffusion (17)(18)(19). However, to distinguish the schemes, it is necessary to vary not only the length of the DNA but also the affinity of the protein for nonspecific DNA (8,11), the latter normally being implemented by varying the salt concentration (20).…”

Requirements for noncovalent binding of vaccinia topoisomerase I to duplex DNA

“…Two possibilities are: (i) topoisomerase binds directly to the CCCTT motif (or related congeners), or (ii) topoisomerase binds initially to duplex DNA without sequence specificity (e.g., through interaction with the DNA backbone) and then diffuses linearly until it encounters a consensus pentamer. The importance of nonspecific DNA protein interactions and facilitated diffusion during site-specific strand scission has been described in a series of elegant studies of the EcoRI endonuclease by Modrich and colleagues (15)(16)(17). Given that the vaccinia topoisomerase can indeed bind nonspecifically, and because potential nonspecific binding sites in natural DNAs (including vaccinia genomic DNA)…”

Stochastic properties of processive cytidine DNA deaminases AID and APOBEC3G

“…Common attributes are that the enzyme has a starting point, the primer/template junction, and then sequentially moves in one direction inserting nucleotides opposite template DNA. Numerous other enzymes use three-dimensional movement by facilitated diffusion, such as DNA glycosylases (Higley & Lloyd 1993;Bennett et al 1995), human AP endonuclease (Carey & Strauss 1999), T4 endonuclease V (Dowd & Lloyd 1990), restriction endonucleases (Jack et al 1982;Terry et al 1985;Stanford et al 2000) and AID and APOBEC3G (Pham et al 2003(Pham et al , 2007Chelico et al 2006). These enzymes face a formidable challenge of finding 'needle in a haystack' substrate motifs in DNA after an arbitrary starting point is established by an initial random binding event.…”