Affinity purification of bacteriophage T4 proteins essential for DNA replication and genetic recombination.

Abstract

The bacteriophage T4 helix-destabilizing protein, the product of gene 32, has been immobilized on an agarose matrix and used for affinity chromatography of lysates of T4-infected Escherichia coli cells. At least 10 T4-encoded early proteins and 3 or 4 host proteins are specifically retained by this gene 32 protein column. Nine of the T4 proteins have been identified as being involved in either DNA replication or genetic recombination. Notably, the T4 DNA polymerase (gene 43 protein) and two major proteins in the recombination pathway (the products of genes uvsX and uvsY) are specifically bound. On a preparative scale, the column is useful for purification of the bound proteins.Gene 32 of bacteriophage T4 encodes a helix-destabilizing protein that is known to be required for the processes of T4 DNA replication, recombination, and repair (1). The purified gene 32 protein has been shown to be an essential component of the T4 in vitro DNA replication system that has been reconstituted from purified proteins (2, 3). Many of the protein-protein interactions involved in bacteriophage DNA metabolism appear to be relatively weak ones. Moreover, the results to be described reveal that a large number of different proteins are involved. For these reasons, we have undertaken the task of cataloguing the protein-protein interactions involving gene 32 protein by protein-affinity chromatography. This approach has several advantages: (i) high concentrations of immobilized proteins can be used, and the resulting high concentration of binding sites on affinity columns makes even weak protein-protein interactions detectable; (ii) both crude lysates and purified proteins can be tested for their binding to such matrices, allowing us both to examine known proteins for their binding to gene 32 protein and to search for unknown ones; and (iii) the method can be used either analytically or preparatively, facilitating large-scale purification of those proteins that bind to the column.As part of our goal of understanding the detailed structure of the T4 DNA replication apparatus, we have used protein affinity chromatography to detect interactions between various different T4 replication proteins (7). Here we describe the associations detected between gene 32 protein and the other proteins present in a T4 bacteriophage-infected cell at early times of infection, when the central genetic processes of DNA replication, recombination, transcription, and repair are underway. This approach has allowed us to identify and to purify several proteins known to be involved in genetic recombination, and it has also given us a new tool for the purification of the T4 DNA polymerase. Greenblatt (8) has described a similar approach in the study of the function of the gene N protein of bacteriophage A. METHODSBuffers. Column buffer was 20 mM Tris-HCl, pH 8.1/1 mM Na3EDTA/1 mM 2-mercaptoethanol/5 mM MgCl2/10% (wt/ vol) glycerol; it was supplemented with 0.05, 0.2, 0.6, or 2.0 M NaCl as noted. Lysis buffer consisted of column buffer supplemented with 50...

Replication Slippage of Different DNA Polymerases Is Inversely Related to Their Strand Displacement Efficiency

“…10C). In contrast, gp32, which stimulates specifically DNA synthesis catalyzed by T4 pol (31,51,60), endows the polymerase with a strand displacement activity, in agreement with previous reports (41,53), probably by direct protein-protein interactions (23,51,52) cific protein-protein interactions, and differences in their processivity. A low processivity would favor heteroduplex formation by promoting the dissociation of the polymerase during replication of the first repeat, whereas high processivity would favor synthesis of parental molecules by preventing the dissociation of the polymerase.…”

UvsX Recombinase and Dda Helicase Rescue Stalled Bacteriophage T4 DNA Replication Forks in Vitro

“…Gene 59 encodes the gp41 helicase loader (27), and when the dda null mutation is combined with a gene 59 amber mutation, DNA synthesis is blocked (22). Most importantly, Dda interacts with UvsX; Dda is retained by a UvsX affinity column (23), and it stimulates UvsX-mediated strand exchange (19). We therefore inquired as to whether the T4 Dda helicase might promote template switching catalyzed by UvsX.…”

“…When dda is combined with an amber mutation in gene 59, which encodes the gp41 helicase loader, the double mutant cannot synthesize DNA under non-permissive conditions (22). Dda interacts both functionally and physically with UvsX (19,23).…”

Biochemical Characterization of Interactions between DNA Polymerase and Single-stranded DNA-binding Protein in Bacteriophage RB69

“…In bacteriophage T4, specific association of gp32 with gp43 has been qualitatively demonstrated using affinity chromatography of radiolabeled crude extracts (17,18). Limited proteolysis has shown that the T4/RB69 gp32 can be divided into three distinct domains.…”