Translocation and specific cleavage of bacteriophage T7 DNA in vivo by Eco KI

Abstract

Infection of Escherichia coli containing the type I restriction enzymeEcoKI by bacteriophage T7 0.3 mutants leads to restriction during the late stages of genome entry and during DNA replication. Patterns of cleavage in vivo suggest that some cutting occurs near the midpoint of two recognition sites, consistent with the idea that EcoKI translocates DNA bidirectionally through itself and cuts when two enzyme molecules collide. Rapid ejection of a 0.3 ؉ T7 genome from a bacteriophage particle results in degradation of the infecting DNA by EcoKI, showing that the normal T7 DNA translocation process delays restriction. A unique recognition site inserted at the genomic left end allows EcoKI to function as a molecular motor and to translocate the remaining 39 kilobases of T7 DNA into the cell.T ype I restriction-modification systems of Escherichia coli serve to protect the bacterium from many phages (1). E. coli K-12 strains harboring the hsd locus contain the enzyme EcoKI, a multisubunit enzyme consisting of a specificity subunit and two each of restriction and modification subunits (2). EcoKI binds S-adenosylmethionine forming an activated enzyme (3), which binds the DNA sequence 5Ј-AACN 6 GTGC (4) and determines its methylation state. If the recognition sequence is nonmethylated, EcoKI hydrolyzes ATP and spools flanking DNA through itself (5); because it remains bound to its recognition sequence, highly twisted DNA loops are formed (6). A current model for cleavage site selection, determined from in vitro experiments, posits that cutting occurs when two translocating EcoKI molecules collide (7). The observation that mutants containing two recognition sites for EcoKI are restricted 15-fold more efficiently than expected if two enzyme molecules cut independently provides in vivo support for the model (8).Many phages and conjugal plasmids escape restriction by type I restriction-modification systems (1, 9). Some phage genomes contain exotically modified bases (10); some plasmids and the phages T7 and T3 synthesize a direct inhibitor of type I restriction-modification systems (11-13). Inhibition of type I restriction by T7 requires stoichiometric amounts of the early protein gp0.3, which binds to EcoKI and prevents it from binding target DNA (14); gp0.3 also obstructs DNA cleavage and methylation after EcoKI has interacted with its recognition sequence (15). Escape of T7 DNA from EcoKI restriction in vivo depends on the synthesis of sufficient gp0.3 before the viral genome becomes cleaved. T7 0.3 mutants containing unmodified DNA plate on hosts harboring EcoKI at an efficiency of 10 Ϫ2 to 10 Ϫ4 (12), somewhat higher but similar to the efficiency of unmodified . Nevertheless, stocks of T7 0.3 mutants can be made on restricting cells, although lysates have low titers and progeny genomes are modified only partially. It was suggested that the incomplete restriction and modification of T7 0.3 DNA was caused by the rapid replication phase and short latent period of the phage.Cellular internalization of T7 DNA is coupled to t...

EcoR124I: from Plasmid-Encoded Restriction-Modification System to Nanodevice

“…While it is far from clear what role the periplasmic exposure of the R-M enzyme would achieve, localization to the cell periphery may hasten endonuclease activity, guarding against incoming DNA. This is to some extent supported by the observation that EcoKI can act as a molecular motor, binding to the first 850 bp of injected DNA and translocating bacteriophage T7 DNA into the cell (26), suggesting localization of the enzyme at the periphery of the cell. …”

Type I Restriction Systems: Sophisticated Molecular Machines (a Legacy of Bertani and Weigle)

“…These proteins, however, are all deficient in ATPase activity and DNA translocation, consistent with a role for the DEAD-box motifs in the coupling of ATP hydrolysis to DNA translocation (35,37). The failure to translocate DNA was demonstrated by an in vivo assay in which wild-type EcoKI translocates the T7 genome from the phage particle into the bacterial cell (57).…”

“…In these experiments, EcoKI bound to two DNA target sites was seen to dimerize prior to the addition of ATP and the initiation of translocation. DNA-dependent dimerization cannot be an absolute requirement for DNA translocation, since a single target is sufficient for DNA translocation (57,77). Dimerization of bound enzymes, however, could facilitate cooperation between two complexes, thereby enhancing restriction.…”

“…The translocation activity of EcoKI assayed by transfer of the T7 genome from the phage particle into the bacterial cell was estimated as ϳ100 bp of DNA per s (37,57). The DEADbox motifs are required for DNA translocation and appear to be organized within domain structures like those deduced for DNA helicases (36).…”

Evidence for DNA Translocation by the ISWI Chromatin-Remodeling Enzyme

“…An assortment of assays have been used to obtain evidence that the DNA translocating subunits of type I restriction enzymes can translocate along DNA (18,22,23,27,37,48,59,63). Also of particular interest is recent evidence suggesting that the yeast Mot1 and Rad54 proteins, which are more closely related to the ATP-dependent chromatin-remodeling enzymes, are able to translocate along DNA (16,50).…”