Translocation and specific cleavage of bacteriophage T7 DNA in vivo by EcoKI

Abstract

Infection of Escherichia coli containing the type I restriction enzyme EcoKI 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 lambda 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.

Figure 1

Autoradiogram depicting the time course of methylation of D364 DNA during an infection, at a multiplicity of 0.4, of the Δhsd recD strain IJ1190 and the hsd⁺ recD strain IJ1409, both containing the dam plasmid pTP166. Time (in minutes) after infection is shown above the lanes. Lanes marked S contain DNA digested with Sau3AI; DNA in other lanes was digested with DpnI. Sau3AI or DpnI fragments are indicated by letters at the left of the panel, and their location on the phage genome is shown on the map below; nucleotide numbers reflect the 578-bp deletion in D364 DNA. The DpnI fragments D+E and F+C arise when the GATC sequences that separate the D and E or F and C fragments are not methylated completely in vivo (18). Arrows at the right of the panel point to bands unique to the infection of IJ1409; fragment sizes were estimated by using a commercial DNA ladder (not shown) and by D364 DNA DpnI fragments. EcoKI recognition sites are shown as triangles, arrows reflect the orientation of the asymmetric EcoKI recognition sequence 5′-AACN₆GTGC, with respect to the sense, or l strand of T7 DNA. To visualize clearly the methylation profile of DNA before and during replication on the same autoradiogram, only 1/10 of the total DNA isolated at time points 18, 21, and 24 min was loaded on the agarose gel. Schematic diagrams of D364 DNA depict GATC sites, EcoKI sites, and predicted sites of EcoKI cleavage (ovals). Predicted DNA fragments (dashed lines) and their approximate sizes (in kilobases) generated by the combination of cleavage at the midpoint between two EcoKI recognition sites and by DpnI digestion are shown at the bottom.

Figure 2

Autoradiogram depicting the time course of methylation of T7 DNA ejected from a λ virion during the infection of the hsd⁺ strain IJ891(pTP166) and of the Δhsd strain IJ1133(pTP166). Time (in minutes) after infection is shown above the lanes. T71.7∷λcosΔ10-NB1[λ] was added to cells at a multiplicity of 0.4. The lane marked Sau3AI illustrates the pattern of cleavage when each GATC site in the phage genome is cut (GATC sites are present in the λ cos DNA; any circular form of intracellular T7 DNA caused by annealing and ligation of the single-stranded ends of T7[λ] DNA cannot be detected in this experiment). DNA in other lanes was cleaved by DpnI; fragments are indicated by letters to the left of the panel, and their location on the phage genome is indicated on the map below; Dam methylation sites (vertical bars) and EcoKI recognition sites (triangles) are also shown. The 103-bp G fragment is not indicated.

Figure 3

Autoradiogram depicting the time course of methylation of sRK836sK0 DNA during the infection of the hsd⁺ strain IJ891(pTP166) and of the isogenic Δhsd strain IJ1133(pTP166). Cells were pretreated with rifampin and infected at a multiplicity of 0.4. Lanes marked S correspond to sRK836sK0 DNA digested with Sau3AI and illustrate the cleavage pattern when every GATC site in the genome is cut. Time (in minutes) after infection is shown above the lanes. DpnI fragments are indicated by letters to the left, and their location on the phage genome is shown on the map below the panel; the single EcoKI recognition site (triangle) is also indicated. The 103-bp G fragment is not indicated.