Association of Holliday-structure resolving endonuclease VII with gp20 from the packaging machine of phage T4

Endonuclease VII (endo VII) is the product of gene 49 (gp49) of bacteriopha ge T4. It is a Holliday-structure resolvase (X-solvase) responsible for cle aring branched replicative DNA prior to packaging. Consequently, mutations in,gene 49 are unable to fill heads to completion because unresolved branch es stop translocation of DNA. A likely association of gp49 with heads or pr oheads, however, could not be shown in the past. We have investigated wheth er gp49 could be part of the transiently assembled packaging machine (the " packasome") located at the base of proheads. Using purified proteins gp16, gp17 and gp20, which are constituents of the packasome, we found that gp49 binds tightly to gp20 and does not bind to gp16 or gp17. Quantification rev ealed that one dimer of gp49 binds one monomer of gp20. Notably, dimerisati on of gp49 was an essential prerequisite for complex formation with gp20, a nd the dimerisation-deficient point mutation His-EVII-W87R showed only resi dual affinity to gp20. Furthermore, truncated peptides of gp49 deficient in dimer formation to various degrees were found to be impaired in binding to gp20. In contrast, the cleavage-deficient mutation EVII-N62D bound normall y to gp20. The cruciform DNA (cf-DNA) resolving activity typical of endo VI I is maintained in gp20-gp49 complexes. Furthermore, the complexes bind cf- DNA in the absence of Mg2+ as demonstrated by electromobility shift assays. The binding of the complexes to cf-DNA occurs ain gp49, since gp20 alone d oes not bind cf-DNA. In conclusion, these findings are consistent with a mo del in which gp49 is an integral part of the packaging machine of phage T4. (C) 1999 Academic Press.