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

Proc. Natl. Acad. Sci. U.S.A.

et al. 2014

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Replication of DNA plays a central role in transmitting hereditary information from cell to cell. To achieve reliable DNA replication, multiple proteins form a stable complex, known as the replisome, enabling them to act together in a highly coordinated fashion. Over the past decade, the roles of the various proteins within the replisome have been determined. Although many of their interactions have been characterized, it remains poorly understood how replication proteins enter and leave the replisome. In this study, we visualize fluorescently labeled bacteriophage T7 DNA polymerases within the replisome while we simultaneously observe the kinetics of the replication process. This combination of observables allows us to monitor both the activity and dynamics of individual polymerases during coordinated leading-and lagging-strand synthesis. Our data suggest that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis and that two or more polymerases are present in the replisome during DNA replication. Our studies imply a highly dynamic picture of the replisome with lagging-strand DNA polymerases residing at the fork for the synthesis of only a few Okazaki fragments. Further, new lagging-strand polymerases are readily recruited from a pool of polymerases that are proximally bound to the replisome and continuously replenished from solution.polymerase exchange | single molecule | fluorescence microscopy T he organization of replisomes is highly conserved among various organisms (1), underlining the evolutionary importance of the replication machinery architecture. The bacteriophage T7 replication system offers an attractive model system to study the interplay between replication proteins because its replication machinery is relatively simple; a functional replisome can be reconstituted by just four purified proteins. Three of these proteins are encoded by the phage itself: helicase-primase (gp4), DNA polymerase (gp5), and single-stranded DNA (ssDNA) binding protein (gp2.5). A processivity factor for the gp5 polymerase, thioredoxin (trx), is provided by the host Escherichia coli.

Section: Methodsmentioning

confidence: 99%

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

Geertsema

Kulczyk

Proc. Natl. Acad. Sci. U.S.A.

et al. 2014

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“…1A). Our earlier studies using gel shift analysis have shown that 1 M gp2.5 is sufficient to coat 3.3 nM 70-mer ssDNA (21). In this experiment containing 4 nM M13 ssDNA, we used a gp2.5 concentration of 20 M, which is sufficient to coat 20 nM M13 ssDNA based on the assumption that one gp2.5 molecule coats 6 -7 nucleotides on ssDNA (8).…”

Section: Effect Of Gp25 and E Coli Ssb Protein On Primermentioning

confidence: 99%

“…Whereas studies with these altered proteins led to the identification of sites that affected DNA binding (7,21) and homologous base pairing (11), none of the altered proteins appeared to be defective in interactions with gp4 or T7 DNA polymerase. One altered protein, gp2.5-F232L, identified in the study, had a single amino acid substitution in the C-terminal residue (20).…”

mentioning

confidence: 99%

Effect of Single-stranded DNA-binding Proteins on the Helicase and Primase Activities of the Bacteriophage T7 Gene 4 Protein

Journal of Biological Chemistry

2004

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Gene 4 protein (gp4) of bacteriophage T7 provides two essential functions at the T7 replication fork, primase and helicase activities. Previous studies have shown that the single-stranded DNA-binding protein of T7, encoded by gene 2.5, interacts with gp4 and modulates its multiple functions. To further characterize the interactions between gp4 and gene 2.5 protein (gp2.5), we have examined the effect of wild-type and altered gene 2.5 proteins as well as Escherichia coli single-stranded DNA-binding (SSB) protein on the ability of gp4 to synthesize primers, hydrolyze dTTP, and unwind duplex DNA. Wild-type gp2.5 and E. coli SSB protein stimulate primer synthesis and DNA-unwinding activities of gp4 at low concentrations but do not significantly affect single-stranded DNA-dependent hydrolysis of dTTP. Neither protein inhibits the binding of gp4 to singlestranded DNA. The variant gene 2.5 proteins, gp2.5-F232L and gp2.5-⌬26C, inhibit primase, dTTPase, and helicase activities proportional to their increased affinities for DNA. Interestingly, wild-type gp2.5 stimulates the unwinding activity of gp4 except at very high concentrations, whereas E. coli SSB protein is highly inhibitory at relative low concentrations.

“…gp2.5 is critical for coordinating leading-and lagging-strand DNA synthesis (22)(23)(24). In addition to DNA binding (25), it interacts with T7 DNA polymerase and gp4 through its acidic C-terminal tail (26,27). However, the effect of gp2.5 on primer synthesis by gp4 has thus far been equivocal (28,29).…”

Section: Primer Release Is Rate Limiting As Determined By Pre-steady-mentioning

confidence: 99%

Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome

Hernandez

Lee

Proc. Natl. Acad. Sci. U.S.A.

2016

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DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading-and lagging-strand DNA synthesis.Okazaki fragment | DNA primase | replisome | primer