Association of Holliday-structure Resolving Endonuclease VII with gp20 from the Packaging Machine of Phage T4

doi:10.1006/jmbi.1998.2399

Journal of Molecular Biology

Volume 285, Issue 3, 22 January 1999, Pages 1131-1144

https://doi.org/10.1006/jmbi.1998.2399 Get rights and content

Abstract

Endonuclease VII (endo VII) is the product of gene 49 (gp49) of bacteriophage T4. It is a Holliday-structure resolvase (X-solvase) responsible for clearing branched replicative DNA prior to packaging. Consequently, mutations in gene 49 are unable to fill heads to completion because unresolved branches stop translocation of DNA. A likely association of gp49 with heads or proheads, however, could not be shown in the past. We have investigated whether gp49 could be part of the transiently assembled packaging machine (the “packasome”) located at the base of proheads. Using purified proteins gpl6, gpl7 and gp20, which are constituents of the packasome, we found that gp49 binds tightly to gp20 and does not bind to gpl6 or gpl7. Quantification revealed that one dimer of gp49 binds one monomer of gp20. Notably, dimerisation of gp49 was an essential prerequisite for complex formation with gp20, and the dimerisation-deficient point mutation His-EVII-W87R showed only residual 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 normally to gp20. The cruciform DNA (cf-DNA) resolving activity typical of endo VII 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 via gp49, since gp20 alone does not bind cf-DNA. In conclusion, these findings are consistent with a model in which gp49 is an integral part of the packaging machine of phage T4.

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^☆: Abbreviations used: endo, endonuclease; cf-, cruciform; ss-, single-stranded; ds-, double-stranded; BSA, bovine serum albumin; EMSA, electromobility shift assay

^☆☆: Edited by J. Karn

^f1: Corresponding author

^f2: E-mail address of the corresponding author: [email protected].

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Journal of Molecular Biology

Regular ArticleAssociation of Holliday-structure Resolving Endonuclease VII with gp20 from the Packaging Machine of Phage T4☆,☆☆

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Endonuclease VII has two DNA-binding sites each composed from one N- and one C-terminus provided by different subunits of the protein dimer

EMBO J.

In vitro processing of heteroduplex loops and mismatches by endonuclease VII

DNA Res.

Inhibition of Holliday structure resolving endonuclease VII of bacteriophage T4 by recombination enzymes UvsX and UvsY

J. Mol. Biol.

DNA packaging in dsDNA bacteriophages

Annu. Rev. Microbiol.

DNA packaging and cutting by phage terminases: control in phage T4 by a synaptic mechanism

BioEssays

Regular Article
Association of Holliday-structure Resolving Endonuclease VII with gp20 from the Packaging Machine of Phage T4☆,☆☆