An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

Kulczyk, Arkadiusz W.; Akabayov, Barak; Lee, Seung-Joo; Bostina, Mihnea; Berkowitz, Steven A.; Richardson, Charles C.

doi:10.1074/jbc.m112.410647

Cited by 28 publications

(44 citation statements)

References 45 publications

(63 reference statements)

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“…When DNA is not present, no binding between gp4-E343Q and gp5/trx is detected, indicating that DNA is required for assembly of the functional gp4-E343Q-gp5/trx complex. In the absence of DNA, ∼70% helicase rings are heptameric, and ∼20% are hexameric, consistent with previous negative-stain EM analysis (5-8), native gel electrophoresis (5,6,21), and gel-filtration data (9).…”

Section: Significancesupporting

confidence: 73%

“…A stable replication complex of gp4-E343Q and gp5 is formed with K d of 0.2 ± 0.04 μM, consistent with fluorescence anisotropy measurements. We have previously characterized gp4-gp5/trx-DNA interactions on the lagging and leading strand using a variety of biophysical methods (5,18). The fork DNA was designed such that it combined DNA constructs used in these experiments.…”

Section: Significancementioning

confidence: 99%

“…Gp4 forms multiple oligomeric forms (5). The negative stain EM revealed that hexamers and heptamers are the most abundant (∼22% and ∼78%, respectively).…”

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Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

Kulczyk

Moeller

Meyer

et al. 2017

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

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We present a structure of the ∼650-kDa functional replisome of bacteriophage T7 assembled on DNA resembling a replication fork. A structure of the complex consisting of six domains of DNA helicase, five domains of RNA primase, two DNA polymerases, and two thioredoxin (processivity factor) molecules was determined by single-particle cryo-electron microscopy. The two molecules of DNA polymerase adopt a different spatial arrangement at the replication fork, reflecting their roles in leading-and lagging-strand synthesis. The structure, in combination with biochemical data, reveals molecular mechanisms for coordination of leading-and laggingstrand synthesis. Because mechanisms of DNA replication are highly conserved, the observations are relevant to other replication systems.cryo-EM structure | replisome | coordination of leading-and lagging-strands synthesis | DNA replication | DNA polymerase T he reverse polarity and antiparallel nature of the two strands in duplex DNA pose a topological problem for their simultaneous synthesis. The "trombone" model of DNA replication postulates that the lagging strand forms a loop such that the leading-and lagging-strand replication proteins contact one another (1). This replication loop contains a nascent Okazaki fragment and allows for coordination of leading-and laggingstrand synthesis. The bacteriophage T7 replisome has been studied extensively (2). Thus, the macromolecular complex of T7 replication proteins provides an excellent model for structural analysis of a replisome. Notably, the human mitochondrial and the T7 replication systems are similar (3).The phage T7 replisome is relatively simple. Four proteins are sufficient for reconstitution of the functional replisome, yet the assembled replisome recapitulates all of the key features of more complex prokaryotic and eukaryotic systems (2, 4). These proteins are the following: DNA polymerase (gp5) and its processivity factor, Escherichia coli thioredoxin (trx), single-stranded DNA (ssDNA)-binding protein (gp2.5), and the bifunctional DNA primase-helicase (gp4).Gp4 forms multiple oligomeric forms (5). The negative stain EM revealed that hexamers and heptamers are the most abundant (∼22% and ∼78%, respectively). Upon addition of ssDNA, the equilibrium between the two oligomeric forms changes (∼77% protein rings are now hexameric, and ∼18% are heptameric), indicative of DNA binding (6). The hexamer is an enzymatically active form of gp4. It binds to ssDNA, hydrolyzes nucleotides, translocates on ssDNA, and unwinds dsDNA (7-9). In contrast, the heptamer does not bind to ssDNA (6).Crystal structures of all of the individual T7 replication proteins have been determined (10-15). However, to date there is no structural information on the T7 replisome or its subassemblies. Consequently, intermolecular interactions involved in coordination of DNA synthesis have not been identified. We have therefore used cryo-electron microscopy (cryo-EM) and single-particle reconstruction methods to determine a structure of the T7 replisome. ResultsR...

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Section: Significancesupporting

confidence: 73%

Section: Significancementioning

confidence: 99%

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Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

Kulczyk

Moeller

Meyer

et al. 2017

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

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“…These studies suggest that the two binding modes of the polymerase to the helicase provide a molecular switching mechanism. Relatively weak electrostatic interactions between the TBD or the front basic patch of the polymerase and the C terminus of the helicase ensure the presence of a nonsynthesizing polymerase at the replisome, whereas an alternative and more tight interaction locks actively synthesizing polymerases onto the helicase (10,(12)(13)(14). Consequently, the T7 helicase theoretically could bind up to six polymerases corresponding to the six C termini of gp4.…”

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confidence: 99%

“…T7 gp5 forms a complex with trx (7,8) to increase its processivity from a few nucleotides to ∼1,000 nucleotides per binding event (9). Basic regions of the DNA polymerase, located in the thioredoxin-binding domain (TBD) as well as on the front of the polymerase, loosely interact with the acidic C-terminal tails of the gp4 helicase (10)(11)(12), whereas a high affinity interaction with gp4 occurs when the polymerase is in a polymerization mode (13,14). T7 gp2.5 also has an acidic C-terminal tail that interacts with the same basic residues of the polymerase TBD (10,15).…”

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confidence: 99%

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

Geertsema

Kulczyk

Richardson

et al. 2014

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

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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.

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Comparison of Bacterial and Eukaryotic Replisome Components

Yao

O'Donnell

2016

Encyclopedia of Cell Biology

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An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

Cited by 28 publications

References 45 publications

Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

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

Comparison of Bacterial and Eukaryotic Replisome Components

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