Rescue of Bacteriophage T7 DNA Polymerase of Low Processivity by Suppressor Mutations Affecting Gene 3 Endonuclease

Lee, Seung-Joo; Chowdhury, Kajal; Tabor, Stanley; Richardson, Charles C.

doi:10.1128/jvi.00855-09

Cited by 7 publications

(9 citation statements)

References 38 publications

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“…1 B and C). In a previous study, we found that T7 lacking gene 5 cannot grow in cells containing gp5 in which these basic residues were replaced with neutral residues, Asn (16). Despite this detrimental effect on T7 growth, the only defect observed was a threefold decrease in polymerase activity on ssDNA templates and a reduced processivity.…”

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confidence: 99%

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Helicase-DNA polymerase interaction is critical to initiate leading-strand DNA synthesis

Zhang

Lee

Zhu

et al. 2011

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

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Interactions between gene 4 helicase and gene 5 DNA polymerase (gp5) are crucial for leading-strand DNA synthesis mediated by the replisome of bacteriophage T7. Interactions between the two proteins that assure high processivity are known but the interactions essential to initiate the leading-strand DNA synthesis remain unidentified. Replacement of solution-exposed basic residues (K587, K589, R590, and R591) located on the front surface of gp5 with neutral asparagines abolishes the ability of gp5 and the helicase to mediate strand-displacement synthesis. This front basic patch in gp5 contributes to physical interactions with the acidic C-terminal tail of the helicase. Nonetheless, the altered polymerase is able to replace gp5 and continue ongoing strand-displacement synthesis. The results suggest that the interaction between the C-terminal tail of the helicase and the basic patch of gp5 is critical for initiation of strand-displacement synthesis. Multiple interactions of T7 DNA polymerase and helicase coordinate replisome movement.DNA polymerase-helicase interaction | strand-displacement DNA synthesis | T7 bacteriophage | T7 replisome B acteriophage T7 has a simple and efficient DNA replication system whose basic reactions mimic those of more complex replication systems (1). The T7 replisome consists of gene 5 DNA polymerase (gp5), the processivity factor, Escherichia coli thioredoxin (trx), gene 4 helicase-primase (gp4), and gene 2.5 ssDNA binding protein (gp2.5) (Fig. 1A). Gp5 forms a high-affinity complex with trx (gp5/trx) to increase the processivity of nucleotide polymerization (2). The C-terminal helicase domain of gp4 assembles as a hexamer and unwinds dsDNA to produce two ssDNA templates for leading-and lagging-strand gp5/trx. The N-terminal primase domain of gp4 catalyzes the synthesis of tetraribonucleotides that are used as primers for the lagging-strand gp5/trx. This gp5/trx also binds to helicase to form a replication loop containing the nascent Okazaki fragment. Gp2.5 coats the ssDNA to remove secondary structures and it also physically interacts with gp4 and gp5/trx, interactions essential for coordination of leading and lagging-strand synthesis (3).Other DNA replication systems are generally more complicated than the T7 system. In E. coli, at least 13 proteins are required for a functional replisome and eight proteins are required in bacteriophage T4 infected cells (1, 4, 5). The two essential helicase and primase are separate proteins although they must physically interact to properly function (5). The additional proteins include processivity clamps and loading proteins. The existing T7 gp4 has usurped helicase and primase functions. The proofreading exonuclease activity resides within the N-terminal portion of gp5, whereas in the E. coli system it resides within ϵ subunit of the polymerase holoenzyme (5). Trx not only binds to gp5 to increase processivity but it also configures the trx-binding loop in gp5 for the binding of gp2.5 and gp4 (6). An interaction of the C-terminal tail of gp2.5 with...

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“…Another basic patch (K587, K589, R590, and R591) is located at the front side of gp5 facing its movement direction along the ssDNA template (denoted as front basic patch, Fbp) (16) (Fig. 1 B and C).…”

mentioning

confidence: 99%

Helicase-DNA polymerase interaction is critical to initiate leading-strand DNA synthesis

Zhang

Lee

Zhu

et al. 2011

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

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“…5B). Interestingly, similar genetically altered gp3 were found to suppress the phenotype arising from an altered T7 DNA polymerase (27). Thus, any abnormal replication structures seem to be targets for gene 3 endonuclease, perhaps providing a check-point for faithful replication.…”

Section: Discussionmentioning

confidence: 98%

“…Phage growth complementation assay was performed as previously described (27). tRNA aminoacylation assay was performed as previously described (31), except that 20 μM RNA from the complex or E. coli total tRNA, 20 μM [ 3 H] leucine or [ 3 H] arginine (15 Ci/mmol), and 2 μM E. coli aminoacyl-tRNA synthetases were used.…”

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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

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

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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 ...

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Recombination-dependent concatemeric viral DNA replication

et al. 2011

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Rescue of Bacteriophage T7 DNA Polymerase of Low Processivity by Suppressor Mutations Affecting Gene 3 Endonuclease

Cited by 7 publications

References 38 publications

Helicase-DNA polymerase interaction is critical to initiate leading-strand DNA synthesis

Helicase-DNA polymerase interaction is critical to initiate leading-strand DNA synthesis

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

Recombination-dependent concatemeric viral DNA replication

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