Motors, Switches, and Contacts in the Replisome

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

Kulczyk

Moeller

Meyer

et al. 2017

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“…The replication machinery of bacteriophage T7 is among the simplest replication systems (4,5). Only four proteins are required to reconstitute coordinated DNA synthesis in vitro: gene 4 primasehelicase (gp4) unwinds the DNA duplex to provide the template for DNA synthesis.…”

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

Hernandez

Lee

Richardson

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

“…Only three T7 proteins-gene 5 DNA polymerase (gp5), gene 4 primase-helicase (gp4), and gene 2.5 ssDNAbinding protein (gp2.5)-together with host Escherichia coli thioredoxin, are sufficient for leading and lagging strands synthesis in a reconstituted replication system (4). The genes encoding these proteins, together with genes 2 (E. coli RNA polymerase inhibitor), 3 (endonuclease), 3.5 (lysozyme), and 6 (exonuclease) constitute the DNA metabolism class II genes.…”

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“…The primase activity of T7 is located in the N-terminal half of the 63-kDa gp4, whereas the C-terminal half of gene 4 encodes the DNA helicase (4). Like other prokaryotic homologs, the primase domain recognizes a specific sequence (1, 6) 5′-(G/T)(G/T) GTC-3′ in the ssDNA template to catalyze the template-directed synthesis of functional tetraribonucleotides (pppACCA, pppACCC, and pppACAC) that the lagging strand DNA polymerase can use as primers to initiate the synthesis of Okazaki fragments.…”

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confidence: 99%

“…Like other prokaryotic homologs, the primase domain recognizes a specific sequence (1, 6) 5′-(G/T)(G/T) GTC-3′ in the ssDNA template to catalyze the template-directed synthesis of functional tetraribonucleotides (pppACCA, pppACCC, and pppACAC) that the lagging strand DNA polymerase can use as primers to initiate the synthesis of Okazaki fragments. Gene 4 encodes two colinear proteins, the full-length 63-kDa gp4 and a short 56-kDa form in equal amount in vivo from an internal start codon and ribosome-binding site (4). This 56-kDa gp4 has full helicase activity but lacks the N-terminal zinc-binding domain (ZBD), an indispensable motif for primer synthesis (7).…”

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Gene 5.5 protein of bacteriophage T7 in complex with Escherichia coli nucleoid protein H-NS and transfer RNA masks transfer RNA priming in T7 DNA replication

Zhu

Lee

Tan

et al. 2012

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DNA primases provide oligoribonucleotides for DNA polymerase to initiate lagging strand synthesis. A deficiency in the primase of bacteriophage T7 to synthesize primers can be overcome by genetic alterations that decrease the expression of T7 gene 5.5, suggesting an alternative mechanism to prime DNA synthesis. The product of gene 5.5 (gp5.5) forms a stable complex with the Escherichia coli histone-like protein H-NS and transfer RNAs (tRNAs). The 3′-terminal sequence (5′-ACCA-3′) of tRNAs is identical to that of a functional primer synthesized by T7 primase. Mutations in T7 that suppress the inability of primase reduce the amount of gp5.5 and thus increase the pool of tRNA to serve as primers. Alterations in T7 gene 3 facilitate tRNA priming by reducing its endonuclease activity that cleaves at the tRNA-DNA junction. The tRNA bound to gp5.5 recruits H-NS. H-NS alone inhibits reactions involved in DNA replication, but the binding to gp5.5-tRNA complex abolishes this inhibition.A 3′-hydroxyl end of an RNA or DNA strand positioned at the catalytic site of DNA polymerase functions as a primer to initiate synthesis of DNA. In most instances, primers are short oligoribonucleotides synthesized by enzymes designated DNA primases (1). DNA primases catalyze the template-directed synthesis of short oligoribonucleotides and stabilize the newly synthesized primer on the template and hand it off to DNA polymerase for initiation of DNA synthesis (2, 3).Bacteriophage T7 has provided a model system for the study of DNA replication. Only three T7 proteins-gene 5 DNA polymerase (gp5), gene 4 primase-helicase (gp4), and gene 2.5 ssDNAbinding protein (gp2.5)-together with host Escherichia coli thioredoxin, are sufficient for leading and lagging strands synthesis in a reconstituted replication system (4). The genes encoding these proteins, together with genes 2 (E. coli RNA polymerase inhibitor), 3 (endonuclease), 3.5 (lysozyme), and 6 (exonuclease) constitute the DNA metabolism class II genes. More than a dozen additional genes within the class II group remain uncharacterized (5).The primase activity of T7 is located in the N-terminal half of the 63-kDa gp4, whereas the C-terminal half of gene 4 encodes the DNA helicase (4). Like other prokaryotic homologs, the primase domain recognizes a specific sequence (1, 6) 5′-(G/T)(G/T) GTC-3′ in the ssDNA template to catalyze the template-directed synthesis of functional tetraribonucleotides (pppACCA, pppACCC, and pppACAC) that the lagging strand DNA polymerase can use as primers to initiate the synthesis of Okazaki fragments. Gene 4 encodes two colinear proteins, the full-length 63-kDa gp4 and a short 56-kDa form in equal amount in vivo from an internal start codon and ribosome-binding site (4). This 56-kDa gp4 has full helicase activity but lacks the N-terminal zinc-binding domain (ZBD), an indispensable motif for primer synthesis (7). Despite its inability to synthesize template-dependent tetraribonucleotides, the 56-kDa gp4 is capable of stabilizing certain preformed oligonucleotides on ...