One subunit of both type I and type III restriction and modification enzymes contains motifs characteristic of DEAD box proteins, which implies that these enzymes may be DNA helicases. This subunit is essential for restriction, but not modification. The current model for restriction by both types of enzyme postulates that DNA cutting is stimulated when two enzyme complexes bound to neighbouring target sequences meet as the consequence of ATP‐dependent DNA translocation. For type I enzymes, this model is supported by in vitro experiments, but the predicted co‐operative interactions between targets have not been detected by assays that monitor restriction in vivo. The experiments reported here clearly establish the required synergistic effect but, in contrast to earlier experiments, they use Escherichia coli K‐12 strains deficient in the restriction alleviation function associated with the Rac prophage. In bacteria with elevated levels of EcoKI the co‐operative interactions are obscured, consistent with co‐operation between free enzyme and that bound at target sites. We have made changes in three of the motifs characteristic of DEAD box proteins, including motif III, which in RecG is implicated in the migration of Holliday junctions. Conservative changes in each of the three motifs impair restriction.

Sty SBLI using the PILEUP program), the sequence for Sty SBLI is anticipated to initiate at codon 27 if translation begins at the first methionine.In the legend to Fig. 5, the lengths of the open reading frames are in error. The legend is corrected below. Fig. 5.Map of the hsd region of S. enterica serovar blegdam. The numbers below the map identify the ORFs; orf3 (540 codons) is hsdM, orf4 (435 codons) is hsdS, and orf5 (1089 codons) is hsdR. The first methionine codon of orf4 overlaps the penultimate codon of orf3. The orientation of the sequence with respect to serB is indicated by the arrow below the map. The order differs from that in E. coli K-12 and S. enterica serovar typhimurium where hsdR is the closest hsd gene to serB.

The cofilins are members of a protein family that binds monomeric and filamentous actin, severs actin filaments, and increases monomer off-rate from the pointed end. Here, we characterize the cofilin-actin interface. We confirm earlier work suggesting the importance of the lower region of subdomain 1 encompassing the N and C termini (site 1) in cofilin binding. In addition, we report the discovery of a new cofilin binding site (site 2) from residues 112-125 that form a helix toward the upper, rear surface of subdomain 1 in the standard actin orientation (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Nature 347, 37-44). We propose that cofilin binds "behind" one monomer and "in front" of the other longitudinally associated monomer, accounting for the fact that cofilin alters the twist in the actin (McGough, A., Pope, B., Chiu, W., and Weeds, A. (1997) J. Cell Biol. 138, 771-781). The characterization of the cofilin-actin interface will facilitate an understanding of how cofilin severs and depolymerizes filaments and may shed light on the mechanism of the gelsolin family because they share a similar fold with the cofilins (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagiki, F. (1996) Cell 85, 1047-1055).Many motile processes in cells require cyclic polymerization and depolymerization of actin filaments. In cell locomotion for example, actin is polymerized at the leading edge of the cell and is recycled by depolymerizing toward the cell center. The rate constants of pure actin have been established (1), and it is clear that a discrepancy exists between these known rates and those calculated from filament turnover in cells (2). A host of actinbinding proteins are known that dramatically alter the behavior of actin in vitro, and of these, the cofilins have been suggested to have the correct properties to increase filament turnover in cells (3). This view has been confirmed by studies with living Saccharomyces (4) and Dictyostelium (5) and by Listeria motility assays (6, 7).The cofilins are a group of low molecular mass (15-21 kDa), actin-binding proteins that depolymerize actin filaments (8).This group includes vertebrate cofilin (9) and ADF 1 (10), twinstar from Drosophila (11), depactin from echinoderms (12), ADFs from plants (13), Unc-60 from nematode (14), cofilins from Saccharomyces (15, 16) and Dictyostelium (17), and actophorin from Acanthamoeba castellanii (18).The mechanism by which cofilin depolymerizes actin filament has been contentious. Soon after the discovery of the first member of the family (10), several authors suggested that depolymerization occurred through severing (9, 18). Evidence for a severing mechanism later came from videomicroscopy (19,20), but this was later challenged (6) as it was shown (6, 21) that cofilins increased the off-rate from the pointed end of the filament. However, the two opinions are not necessarily exclusive (22-24), and a similar mechanism has been proposed for both events (23). We report the identification of a cof...

The type I restriction and modification (R-M) enzyme from Salmonella enterica serovar kaduna ( Sty SKI) recognises the DNA sequence 5'-CGAT(N)7GTTA, an unusual target for a type I R-M system in that it comprises two tetranucleotide components. The amino target recognition domain (TRD) of Sty SKI recognises 5'-CGAT and shows 36% amino acid identity with the carboxy TRD of Eco R124I which recognises the complementary, but degenerate, sequence 5'-RTCG. Current models predict that the amino and carboxy TRDs of the specificity subunit are in inverted orientations within a structure with 2-fold rotational symmetry. The complementary target sequences recognised by the amino TRD of Sty SKI and the carboxy TRD of Eco R124I are consistent with the predicted inverted positions of the TRDs. Amino TRDs of similar amino acid sequence have been shown to recognise the same nucleotide sequence. The similarity reported here, the first example of one between amino and carboxy TRDs, while consistent with a conserved mechanism of target recognition, offers additional flexibility in the evolution of sequence specificity by increasing the potential diversity of DNA targets for a given number of TRDs. Sty SKI identifies the first member of the IB family in Salmonella species.

Cofilin is a small actin-binding protein that is known to bind both F-actin and G-actin, severing the former. The interaction of cofilin with actin is pH-sensitive, F-actin being preferentially bound at low pH and G-actin at higher pH, within the physiological range. Diffusion coefficients of F-actin with cofilin were measured by the fluorescence recovery after photobleaching (FRAP) technique. This has the potential for simultaneous and direct measurement of average polymer length via the average diffusion coefficient of the polymers (D LM ) as well as the fraction of polymerized actin, f LM , present in solution. In the range of cofilin±actin ratios up to 1 : 1 and at both pH 6.5 and pH 8.0, the diffusion coefficients of the polymers increased with the amount of cofilin present in the complex, in a co-operative manner to a plateau. We interpret this as indicating co-operative binding/severing and that filaments less than a certain length cannot be severed further. Under the conditions used here, filaments were found to be more motile at pH 6.5 than at pH 8.0. At pH 8.0, some actin is expected to be sequestered as ADP±actin±cofilin complexes, with the remaining actin being present as long slowly diffusing filaments. At pH 6.5, however, cofilin binds to F-actin to form short rapidly diffusing cofilaments. These filaments form very rapidly from cofilin±actin monomeric complexes, possibly indicating that this complex is able to polymerize without dissociation. These findings may be relevant to the nuclear import of actin±cofilin complexes.