A DNA-Binding Protein Induced by Bacteriophage T7

Abstract

A DNA-binding protein has been purified from Escherichia coli infected with bacteriophage T7 by DNA-cellulose chromatography. The protein is absent in uninfected cells. The purified protein has a molecular weight of 31,000 and binds strongly and preferentially to single-stranded DNA. In vitro studies show that this protein can stimulate the rate of polymerization catalyzed by the T7-induced DNA polymerase 10-15 times under conditions where the polymerase is unable to effectively use a single-stranded template. The degree of stimulation is dependent upon the ratio of binding protein to DNA template and is independent of polymerase concentration. The observed stimulation is specific for the T7 DNA polymerase in that addition of the protein to reactions catalyzed by E. coli DNA polymerases I, II, or III or T4 DNA polymerase is without effect.

The DNA Binding Domain of the Gene 2.5 Single-stranded DNA-binding Protein of Bacteriophage T7

“…We are currently pursuing the mechanism of this reaction by studying another altered protein that binds ssDNA but cannot facilitate the annealing of homologous strands of ssDNA. 3 From the data presented here, we conclude that gp2.5 must be able to bind ssDNA to facilitate DNA annealing.…”

“…For this reason, we feel the lack of annealing we observed is related to the affinity of the altered proteins for ssDNA rather than reflecting a defect in the basic mechanism of homologous base-pairing. Interestingly, gp2.5-⌬26C facilitates strand annealing more efficiently than the wild-type protein, 3 further supporting a relationship between ssDNA binding affinity and homologous base pair annealing. Furthermore, despite its unaltered affinity for ssDNA the variant protein K109I is defective in base pairing, requiring 2-fold more protein to completely anneal all the substrate.…”

“…The interaction is weaker than that observed with T7 DNA polymerase and cannot be detected using surface plasmon resonance. 3 Therefore, we examined the interaction of gp2.5 with gene 4 protein using affinity chromatography. The 63-kDa gene 4 protein binds to a wt gp 2.5 affinity column and elutes over a broad range of salt concentrations (100 to 250 mM NaCl) (data not shown).…”

“…Bacteriophage T7 encodes its own ssDNAbinding protein, the product of gene 2.5. Gene 2.5 protein is essential for phage survival (2) and plays multiple roles in DNA replication, recombination, and repair (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Gene 2.5 protein interacts directly with both the T7 DNA polymerase (9) and the gene 4 helicase/primase (7), stimulating the activity of each protein.…”

Role of the Acidic Carboxyl-terminal Domain of the Single-stranded DNA-binding Protein of Bacteriophage T7 in Specific Protein-Protein Interactions

“…Stimulation of T7 DNA Polymerase-T7 gene 2.5 protein, like E. coli SSB protein and T4 gene 32 protein (10,18,42), stimulates the activity of its cognate DNA polymerase (7,38,(43)(44)(45). T7 gene 2.5-⌬21C on the other hand has no affect on T7 DNA polymerase activity, although it retains its ability to bind to ssDNA (30), a result that is not surprising in view of its inability to interact physically with T7 DNA polymerase as presented above.…”