DNA-Thumb Interactions and Processivity of T7 DNA Polymerase in Comparison to Yeast Polymerase η

Cannistraro, Vincent J.; Taylor, John‐Stephen

doi:10.1074/jbc.m400282200

Cited by 25 publications

(38 citation statements)

References 43 publications

(39 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In contrast to E. coli DNA polymerase III, trx is not known to encircle the DNA as a clamp (24). The crystal structure of gp5/trx in complex with the primer-template is believed to be that of the enzyme in the nonprocessive mode (11).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Interactions of Escherichia coli Thioredoxin, the Processivity Factor, with Bacteriophage T7 DNA Polymerase and Helicase

Ghosh

Hamdan

Cook

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

Escherichia coli thioredoxin binds to a unique flexible loop of 71 amino acid residues, designated the thioredoxin binding domain (TBD), located in the thumb subdomain of bacteriophage T7 gene 5 DNA polymerase. The initial designation of thioredoxin as a processivity factor was premature. Rather it remodels the TBD for interaction with DNA and the other replication proteins. The binding of thioredoxin exposes a number of basic residues on the TBD that lie over the duplex region of the primer-template and increases the processivity of nucleotide polymerization. Two small solvent-exposed loops (loops A and B) located within TBD electrostatically interact with the acidic C-terminal tail of T7 gene 4 helicase-primase, an interaction that is enhanced by the binding of thioredoxin. Several basic residues on the surface of thioredoxin in the polymerase-thioredoxin complex lie in close proximity to the TBD. One of these residues, lysine 36, is located proximal to loop A. The substitution of glutamate for lysine has a dramatic effect on the binding of gene 4 helicase to a DNA polymerase-thioredoxin complex lacking charges on loop B; binding is decreased 15-fold relative to that observed with wild-type thioredoxin. This defective interaction impairs the ability of T7 DNA polymerase-thioredoxin together with T7 helicase to mediate strand displacement synthesis. This is the first demonstration that thioredoxin interacts with replication proteins other than T7 DNA polymerase.Bacteriophage T7 encodes its own replicative DNA polymerase, the product of gene 5 of the phage (1). The gene 5 protein (gp5) is a nonprocessive DNA polymerase, dissociating from the primer-template after the polymerization of 1-20 nucleotides (2). Upon infection of Escherichia coli gp5 forms a one-toone complex with the host thioredoxin (trx) 2 (3). The binding of trx to gp5 increases the processivity to ϳ800 nucleotides per binding event (3, 4). The crystal structure of gp5/trx in complex with a primer-template and a nucleoside triphosphate reveals trx bound to a unique 71-amino acid loop lying between helices H1 and H2 in the thumb subdomain of gp5 (5). This thioredoxin binding domain (TBD) is not present in other members of this family such as E. coli DNA polymerase I and TaqDNA polymerase (5, 6). The largely hydrophobic interaction of trx with gp5 provides for considerable stability (K d ϭ 5 nM) (2). A number of basic residues in the TBD lie over the duplex portion of the primer-template within the DNA binding crevice and presumably account for the increase in processivity via electrostatic interactions (7,8). This requirement of trx as a processivity factor for T7 DNA polymerase provides one explanation for its essential function in T7 growth; T7 does not grow in E. coli cells lacking trx (9, 10).The crystal structure of the complex also reveals two small, solvent-exposed basic loops (loops A and B) in the TBD (Fig. 1A) (5, 11). Recent studies have shown that both T7 gene 4 helicase-primase (gp4) and T7 gene 2.5 ssDNA-binding protein (gp2.5) bind ...

show abstract

Section: Discussionmentioning

confidence: 99%

“…The polymerization stalls when it encounters secondary structures on ssDNA, and this pausing can be prevented by binding of singlestranded DNA proteins (2,12,22). A replisome consisting of gp5/trx, gp4, and gp2.5 mediate coordinated DNA synthesis in vitro, where leading and lagging strand synthesis proceed at identical rates (24).…”

Section: Discussionmentioning

confidence: 99%

Interactions of Escherichia coli Thioredoxin, the Processivity Factor, with Bacteriophage T7 DNA Polymerase and Helicase

Ghosh

Hamdan

Cook

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…2D), so it is possible that this residue is a major determinant of fidelity. A further point of interest is that POLN has an insertion in the tip of the thumb subdomain, shorter than the 22-residue insert of POLQ (5), but in a region of the protein that is important for DNA binding (22,48,49).…”

Section: Discussionmentioning

confidence: 99%

Human DNA Polymerase N (POLN) Is a Low Fidelity Enzyme Capable of Error-free Bypass of 5S-Thymine Glycol

Takata

Shimizu

Iwai

et al. 2006

Journal of Biological Chemistry

139

206

View full text Add to dashboard Cite

Human DNA polymerase N (POLN or pol ) is the most recently discovered nuclear DNA polymerase in the human genome. It is an A-family DNA polymerase related to Escherichia coli pol I, human POLQ, and Drosophila Mus308. We report the first purification of the recombinant enzyme and examination of its biochemical properties, as a step toward understanding the functions of POLN. Unusual for an A-family DNA polymerase, POLN is a low fidelity enzyme incorporating T opposite template G with a frequency of 0.45 and G opposite template T with a frequency of 0.021. The frequency of misincorporation of T opposite template G is higher than any other known DNA polymerase. POLN has a processivity of DNA synthesis (1-100 nucleotides) similar to the exonuclease-deficient Klenow fragment of E. coli pol I, is inhibited by dideoxynucleotides, and resistant to aphidicolin. The strand displacement activity of POLN was higher than exonuclease-deficient Klenow fragment. Furthermore, POLN can perform translesion synthesis past thymine glycol, a common endogenous and radiationinduced product of reactive oxygen species damage to DNA. Thymine glycol blocks DNA synthesis by most DNA polymerases, but POLN was particularly adept at efficient and accurate translesion synthesis past a 5S-thymine glycol.The human genome contains 15 distinct known DNA polymerase genes, and these are classified into four families A, B, X, and Y based on their amino acid sequences (1). Human DNA polymerase N (POLN), 2 the most recently discovered nuclear DNA polymerase, is an A-family enzyme with unknown function. The gene on chromosome 4p16.2 encodes a protein of 900 amino acid residues with a molecular mass of 100 kDa (2). The prototypical A-family DNA polymerase, Escherichia coli pol I is a high fidelity DNA polymerase that contributes to the maturation of Okazaki fragments during DNA replication and in gap-filling during base excision repair (BER), nucleotide excision repair (NER), and repair of DNA interstrand cross-links.Human POLQ, another A-family DNA polymerase, is similar to the Drosophila nuclear DNA polymerase Mus308 (3) in that it encodes both a DNA/RNA helicase domain and an A-family DNA polymerase domain (4, 5). By contrast, POLN has only the DNA polymerase domain. The POLN gene is encoded only in vertebrate genomes, but not in invertebrates or any lower eukaryotes. Possibly POLN has a role related to organ systems that are especially developed in vertebrates, such as the adaptive immune system or the brain. Expression studies of POLN are limited, but expression of the gene has been detected by Northern blotting in testes, heart and skeletal muscle tissue (2) and by expression sequence tagging in prostate, muscle, brain, and other organs. In cells from a human neuroblastoma patient, a chromosome fusion (1, 4) disrupting the DNA polymerase domain coding sequence of POLN was observed at diagnosis and at relapse. A (4, 17) fusion was detected at relapse only (6). It is possible that POLN might serve as a tumor suppressor in some cell types and that loss...

show abstract

“…Recent measurements of the processivity of gp5/trx by using single molecule techniques yield values from 700 to 1,000 nucleotides per binding event (22,23). Earlier ensemble experiments used to measure processivity have mainly relied on dilution or the addition of a DNA ''trap'' to limit the observed DNA synthesis to a single binding event (24,25). However, the availability of gp5-Y526F/trx provides an additional method for measuring processivity.…”

Section: T7mentioning

confidence: 99%

Exchange of DNA polymerases at the replication fork of bacteriophage T7

Johnson

Takahashi

Hamdan

et al. 2007

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

View full text Add to dashboard Cite

T7 gene 5 DNA polymerase (gp5) and its processivity factor, Escherichia coli thioredoxin, together with the T7 gene 4 DNA helicase, catalyze strand displacement synthesis on duplex DNA processively (>17,000 nucleotides per binding event). The processive DNA synthesis is resistant to the addition of a DNA trap. However, when the polymerase-thioredoxin complex actively synthesizing DNA is challenged with excess DNA polymerase-thioredoxin exchange occurs readily. The exchange can be monitored by the use of a genetically altered T7 DNA polymerase (gp5-Y526F) in which tyrosine-526 is replaced with phenylalanine. DNA synthesis catalyzed by gp5-Y526F is resistant to inhibition by chain-terminating dideoxynucleotides because gp5-Y526F is deficient in the incorporation of these analogs relative to the wild-type enzyme. The exchange also occurs during coordinated DNA synthesis in which leading-and lagging-strand synthesis occur at the same rate. On ssDNA templates with the T7 DNA polymerase alone, such exchange is not evident, suggesting that free polymerase is first recruited to the replisome by means of T7 gene 4 helicase. The ability to exchange DNA polymerases within the replisome without affecting processivity provides advantages for fidelity as well as the cycling of the polymerase from a completed Okazaki fragment to a new primer on the lagging strand.processivity ͉ replisome ͉ gp5-gp4 ͉ dynamic T he replisome (Fig. 1) of bacteriophage T7 consists of three phage-encoded proteins and one host protein (1). DNA synthesis is carried out by the gene 5 DNA polymerase (gp5) after forming a tight one-to-one complex with Escherichia coli thioredoxin (trx), the processivity factor (2, 3). The multifunctional gene 4 protein (gp4) provides both primase and helicase activities (4-7), and the gene 2.5 protein (gp2.5) functions as a ssDNA-binding protein (8). Although the size of the T7 replisome is modest, the multiple interactions of the four proteins give rise to functions provided by additional proteins in other systems. For example, a unique insert in the thumb of gp5, the thioredoxin-binding domain (TBD), provides a binding site for the processivity factor (9-11) and for the binding of gp4 and gp2.5 (11). Furthermore, the covalent attachment of the primase to the helicase in gp4 provides for the requisite interaction of these two components of the replisome. Accessory proteins such as clamp and helicase loaders are not required in the T7 system. Nonetheless, leading-and lagging-strand syntheses are processive and coordinated (12). The lagging strand is synthesized in a discontinuous manner with nascent Okazaki fragments located in replication loops (12,13).A mechanism to coordinate leading-and lagging-strand syntheses is dictated by the inherently more complex events that occur on the lagging strand. Whereas the leading strand can be synthesized in an uninterrupted fashion, lagging-strand synthesis must occur through the synthesis of short Okazaki fragments (Fig. 1), an event that necessitates the synthesis of an RNA primer...

show abstract

DNA-Thumb Interactions and Processivity of T7 DNA Polymerase in Comparison to Yeast Polymerase η

Cited by 25 publications

References 43 publications

Interactions of Escherichia coli Thioredoxin, the Processivity Factor, with Bacteriophage T7 DNA Polymerase and Helicase

Interactions of Escherichia coli Thioredoxin, the Processivity Factor, with Bacteriophage T7 DNA Polymerase and Helicase

Human DNA Polymerase N (POLN) Is a Low Fidelity Enzyme Capable of Error-free Bypass of 5S-Thymine Glycol

Exchange of DNA polymerases at the replication fork of bacteriophage T7

Contact Info

Product

Resources

About