Accurate Scanning of the BssHII Endonuclease in Search for Its DNA Cleavage Site

Abstract

A facilitated diffusion mechanism has been proposed to account for the kinetic efficiency with which restriction endonucleases are able to locate DNA recognition sites. Such a mechanism involves the initial formation of a nonspecific complex upon collision of the protein with the DNA, with the subsequent diffusion of the protein along the DNA helix until either a recognition site is located or the protein dissociates into solution. Protein translocation may be facilitated by either sliding along the DNA, hopping to nearby sites, or intersegment transfer over larger distances. Previous analyses of the manner in which restriction enzymes cleave DNA substrates did rule out the latter mechanism. To discriminate between protein sliding or scanning and protein hopping, we designed a unique DNA template with three overlapping, mutually exclusive recognition sites for the BssHII endonuclease. Analysis of the cleavage pattern demonstrated efficient usage of both external sites, whereas the centrally located site was not efficiently cleaved. These results confirm that linear diffusion of the BssHII enzyme occurs by scanning along the DNA. Furthermore, the scanning enzyme was found to stop and cleave at the first site encountered. Thus, a sliding restriction endonuclease recognizes cleavage sites with high fidelity, without skipping of potential sites.It is unlikely that site-specific DNA-binding proteins locate their target site by simple three-dimensional diffusion or trialand-error mechanism (reviewed in Ref. 1). In many cases, proteins use a nonspecific DNA binding affinity to remain attached to the DNA, where it moves freely and rapidly along the length of the DNA in search of specific target sequences. This mechanism will reduce the dimensionality of the search process and consequently speed up the rate of target location. Several mechanisms for the facilitated transfer of protein along a DNA ligand have been proposed (2). These include a sliding mechanism along the DNA and several types of dissociation-reassociation processes (e.g. hopping over short distances or intersegment transfer). This translocation reaction is interrupted either by location of and binding to the specific target site or by dissociation of the protein from the DNA molecule. The sliding or scanning mechanism has been observed for several DNAinteracting proteins like the Escherichia coli lac repressor (reviewed in Ref.3), RNA polymerase (4, 5), and Micrococcus luteus UV endonuclease (6).Despite their abundant use in molecular biology, only a few restriction endonucleases have been studied seriously as DNAbinding enzymes. The best understood of the type II restriction enzymes is the EcoRI endonuclease (7-10). Diffusion mechanisms along the DNA helix contour have been proposed to account for the kinetic efficiency with which this protein is able to locate its recognition and cleavage sites. Similar diffusion/ sliding/scanning mechanisms have been proposed for the HindIII and BamHI endonucleases, as well as for the BamHI methylase enzyme (10, 11).Kn...

The Dimer Initiation Site Hairpin Mediates Dimerization of the Human Immunodeficiency Virus, Type 2 RNA Genome

“…We think that the assay conditions used in the other studies favor the formation of DIS-independent RNA interactions, which correspond to the background levels measured in our assay system. Obviously, it remains possible that the TAR and PBS domains act as secondary dimerization sites (26), but our data demonstrate that the DIS motif is critical for the initial RNA-RNA contact and dimerization.…”

“…For instance, whereas the unnatural palindrome GUC-GAC is highly efficient in the HIV-2 DIS, it has been shown to function poorly in an HIV-1 background (38). Likewise, the two inactive palindromes UGUACA and GUUAAC with four A/U nucleotides are found in the DIS of naturally occurring virus isolates of SIV syk and SIV mnd , respectively (26). Assuming that these DIS elements function in RNA dimerization in their natural context, it will be of interest to map putative enhancer elements that allow the use of such A/U-rich palindromes in these viruses.…”

“…The effect of mutations in the DIS hairpin on virus replication is not very severe, rendering viral infectivity somewhat reduced because of diminished packaging of the RNA genome. Remarkably, packaged RNA exists as dimers with a thermal stability that is similar to that of the wild type genome (21,26,27). Relatively minor dimerization defects have been reported in some studies (14,28).…”

“…Although the overall structure of the HIV-2 leader RNA is similar to that of HIV-1 (29), including a DIS hairpin motif with a palindromic loop sequence, controversy remains about the region that is essential for HIV-2 dimer formation. Different studies have suggested an involvement of the transactivation region (TAR) hairpin (9), the primer binding site (PBS) (30), or the DIS hairpin (26,29). We performed a detailed mutational analysis to determine which regions are involved in dimerization of the HIV-2 genome.…”

The Kissing Hairpin Sequence Promotes Recombination within the HIV-I 5′ Leader Region

“…The viral nucleocapsid protein, although dispensable for RNA dimerization, can enhance the process (46,49). The DIS hairpin is dispensable for virus replication, although deletion of the element is associated with reduced encapsidation, minus strand synthesis, and infectivity, as well as altered replication kinetics (50,51). In addition to the DIS hairpin, the HIV-I 5Ј leader sequence encodes multiple cis-acting elements, including the packaging signal () and splice donor site, that regulate distinct steps in the virus life cycle (52) (Fig.…”