The Carboxyl-terminal Domain of Bacteriophage T7 Single-stranded DNA-binding Protein Modulates DNA Binding and Interaction with T7 DNA Polymerase

Abstract

Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein (gp2.5). Previous studies have demonstrated that the acidic carboxyl terminus of the protein is essential and that it mediates multiple protein-protein interactions. A screen for lethal mutations in gene 2.5 uncovered a variety of essential amino acids, among which was a single amino acid substitution, F232L, at the carboxyl-terminal residue. gp2.5-F232L exhibits a 3-fold increase in binding affinity for single-stranded DNA and a slightly lower affinity for T7 DNA polymerase when compared with wild type gp2.5. gp2.5-F232L stimulates the activity of T7 DNA polymerase and, in contrast to wild-type gp2.5, promotes strand displacement DNA synthesis by T7 DNA polymerase. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-⌬26C, binds single-stranded DNA 40-fold more tightly than the wild-type protein and cannot physically interact with T7 DNA polymerase. gp2.5-⌬26C is inhibitory for DNA synthesis catalyzed by T7 DNA polymerase on single-stranded DNA, and it does not stimulate strand displacement DNA synthesis at high concentration. The biochemical and genetic data support a model in which the carboxyl-terminal tail modulates DNA binding and mediates essential interactions with T7 DNA polymerase.Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA (ssDNA) 1 -binding protein (gp2.5) that is essential for viral survival (1). gp2.5 modulates several important reactions in DNA replication, recombination, and repair (1-12). The fundamental reactions at the T7 phage replication fork can be reconstituted with only four proteins (13, 14): T7 gene 5 DNA polymerase, its processivity factor Escherichia coli thioredoxin (15, 16), T7 gene 4 helicase/primase (17-19), and T7 gp2.5. gp2.5 physically interacts with T7 DNA polymerase and T7 helicase/primase to stimulate their activities (6,8). The binding of gp2.5 to ssDNA is critical because it affects both specific DNA-protein and protein-protein interactions in the replisome (14,20). In this regard it is essential for coupling leading and lagging strand DNA synthesis in vitro (14). gp2.5 is also essential for recombination in T7 phage-infected cells, and in addition to the interactions described above, it also mediates homologous base pairing (11).Despite a lack of sequence homology, T7 gp2.5 is functionally similar to the extensively studied SSB protein of E. coli and the gene 32 protein of bacteriophage T4. Like gp2.5, they are both ssDNA-binding proteins, a class of ubiquitous proteins that are not only essential in DNA replication but also play key roles in DNA recombination and repair (7, 21). Biochemical studies have shown that these proteins, like T7 gp2.5, interact with other proteins at the replication fork. E. coli SSB protein interacts with E. coli DNA polymerase II, exonuclease I, and other proteins involved in replication (22-24). T4 gene 32 protein physically interacts with at least 10 T4-encoded proteins, including T4 DNA polymerase, that are involved in T4...

A Molecular Handoff between Bacteriophage T7 DNA Primase and T7 DNA Polymerase Initiates DNA Synthesis

“…10a, lanes 4, 7, 10, and 13). gp2.5⌬26 binds to ssDNA with 40-fold higher affinity than wild-type gp2.5 (9,53), yet the mutant protein interfered weakly with primer utilization by T7 DNA polymerase alone, and gp2.5⌬26 did not inhibit DNA synthesis in the presence of T7 primase. These results indicate the interaction of T7 DNA polymerase with gp2.5 specifically interferes with DNA synthesis that is initiated from an oligoribonucleotide primer.…”

“…First, gp2.5 strongly inhibited primer utilization in the presence of a vast excess of ssDNA template and tetraribonucleotide primer. Second, a mutant gp2.5 protein (gp2.5⌬26) with decreased binding affinity for T7 DNA polymerase was a less potent inhibitor of primer utilization even though gp2.5⌬26 binds to ssDNA 40-fold more tightly than wild-type gp2.5 (53). Although gp2.5 binds to T7 primase-helicase (9), it is unlikely this interaction involves the region comprising the primase.…”

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

“…The sequences of the major product are shown on the right of the autoradiograph, and the letters r and d represent ribo-and deoxyribonucleotide, respectively. exonuclease activity catalyzes strand displacement synthesis for several hundred nucleotides (23), and the presence of E. coli SSB proteins enables this reaction to occur (26,27). "Breathing" of the 5Ј-terminus of the strand to be displaced most likely precedes the initiation of strand displacement synthesis.…”

“…Whether or not exonuclease-deficient DNA polymerase exists in T7 phage-infected cells is not known. E. coli ssDNA binding protein (SSB protein), which exists abundantly in phage-infected cells, enables gp5 to catalyze strand displacement synthesis (26,27). Finally, T7 gene 2.5 ssDNAbinding protein enables T7 DNA polymerase to catalyze strand displacement synthesis at a nick in duplex DNA sufficient for the loading of T7 DNA helicase (27,28).…”

Acidic C-terminal tail of the ssDNA-binding protein of bacteriophage T7 and ssDNA compete for the same binding surface

“…This fragment also is the site of binding of the processivity factor, E. coli trx, and the C terminus of gene 4 helicase-primase. Thus, the C-terminal tail of gp2.5 could potentially interfere with all interaction pairs at the T7 replication fork: directly by being part of the contact interface itself or indirectly by competing with another partner for the same contact surface (11,23,26,27). The multiple interactions of the C terminus of gp2.5 could thus function as one mechanism to coordinate the multiple reactions occurring at the replication fork.…”