Identification and Mapping of Protein-Protein Interactions between gp32 and gp59 by Cross-linking

Faoud Ishmael, Stephen C. Alley, Stephen Benkovic

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

The bacteriophage T4 59 protein (gp59) plays a vital role in recombination and replication by promoting the assembly of the gene 41 helicase (gp41) onto DNA, thus enabling replication as well as strand exchange in recombination. Loading of the helicase onto gp32 (the T4 single strand binding protein)-coated single-stranded DNA requires gp59 to remove gp32 and replace it with gp41. Cross-linking studies between gp32 and gp59 reveal an interaction between Cys-166 of gp32 and Cys-42 of gp59. Since Cys-166 lies in the DNA binding core domain of gp32, this interaction may affect the association of gp32 with DNA. In the presence of gp32 or DNA, gp59 is capable of forming a multimer consisting of at least five gp59 subunits. Kinetics studies suggest that gp59 and gp41 exist in a one-to-one ratio, predicting that gp59 is capable of forming a hexamer (Raney, K. D., Carver, T. E., and Benkovic, S. J. (1996) J. Biol. Chem. 271, 14074-14081). The C-terminal A-domain of gp32 is needed for gp59 oligomer formation. Cross-linking has established that gp59 can interact with gp32-A (a truncated form of gp32 lacking the A-domain) but cannot form higher species. The results support a model in which gp59 binds to gp32 on a replication fork, destabilizing the gp32-single-stranded DNA interaction concomitant with the oligomerization of gp59 that results in a switching of gp41 for gp32 at the replication fork.

Original languageEnglish (US)
Pages (from-to)25236-25242
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number27
DOIs
StatePublished - Jul 6 2001

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Single-Stranded DNA
DNA
Genetic Recombination
Proteins
Oligomerization
Bacteriophages
DNA-Binding Proteins
Oligomers
Carrier Proteins
Genes
Association reactions
Kinetics
Enterobacteria phage T4 gene 59 protein

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Identification and Mapping of Protein-Protein Interactions between gp32 and gp59 by Cross-linking",
abstract = "The bacteriophage T4 59 protein (gp59) plays a vital role in recombination and replication by promoting the assembly of the gene 41 helicase (gp41) onto DNA, thus enabling replication as well as strand exchange in recombination. Loading of the helicase onto gp32 (the T4 single strand binding protein)-coated single-stranded DNA requires gp59 to remove gp32 and replace it with gp41. Cross-linking studies between gp32 and gp59 reveal an interaction between Cys-166 of gp32 and Cys-42 of gp59. Since Cys-166 lies in the DNA binding core domain of gp32, this interaction may affect the association of gp32 with DNA. In the presence of gp32 or DNA, gp59 is capable of forming a multimer consisting of at least five gp59 subunits. Kinetics studies suggest that gp59 and gp41 exist in a one-to-one ratio, predicting that gp59 is capable of forming a hexamer (Raney, K. D., Carver, T. E., and Benkovic, S. J. (1996) J. Biol. Chem. 271, 14074-14081). The C-terminal A-domain of gp32 is needed for gp59 oligomer formation. Cross-linking has established that gp59 can interact with gp32-A (a truncated form of gp32 lacking the A-domain) but cannot form higher species. The results support a model in which gp59 binds to gp32 on a replication fork, destabilizing the gp32-single-stranded DNA interaction concomitant with the oligomerization of gp59 that results in a switching of gp41 for gp32 at the replication fork.",
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Identification and Mapping of Protein-Protein Interactions between gp32 and gp59 by Cross-linking. / Ishmael, Faoud; Alley, Stephen C.; Benkovic, Stephen.

In: Journal of Biological Chemistry, Vol. 276, No. 27, 06.07.2001, p. 25236-25242.

Research output: Contribution to journalArticle

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N2 - The bacteriophage T4 59 protein (gp59) plays a vital role in recombination and replication by promoting the assembly of the gene 41 helicase (gp41) onto DNA, thus enabling replication as well as strand exchange in recombination. Loading of the helicase onto gp32 (the T4 single strand binding protein)-coated single-stranded DNA requires gp59 to remove gp32 and replace it with gp41. Cross-linking studies between gp32 and gp59 reveal an interaction between Cys-166 of gp32 and Cys-42 of gp59. Since Cys-166 lies in the DNA binding core domain of gp32, this interaction may affect the association of gp32 with DNA. In the presence of gp32 or DNA, gp59 is capable of forming a multimer consisting of at least five gp59 subunits. Kinetics studies suggest that gp59 and gp41 exist in a one-to-one ratio, predicting that gp59 is capable of forming a hexamer (Raney, K. D., Carver, T. E., and Benkovic, S. J. (1996) J. Biol. Chem. 271, 14074-14081). The C-terminal A-domain of gp32 is needed for gp59 oligomer formation. Cross-linking has established that gp59 can interact with gp32-A (a truncated form of gp32 lacking the A-domain) but cannot form higher species. The results support a model in which gp59 binds to gp32 on a replication fork, destabilizing the gp32-single-stranded DNA interaction concomitant with the oligomerization of gp59 that results in a switching of gp41 for gp32 at the replication fork.

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