The Linker Region between the Helicase and Primase Domains of the Gene 4 Protein of Bacteriophage T7

Lee, Seung-Joo; Richardson, Charles C.

doi:10.1074/jbc.m400857200

Cited by 32 publications

(42 citation statements)

References 37 publications

(57 reference statements)

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“…A326 is well conserved between mtDNA helicase and bacteriophage T7 gp4, where the analogous residue is Ala 257 . Although mutants at position Ala 257 in bacteriophage T7 gp4 (A257L, A257P, and A257V) show defects in helicase activity in vitro, they fail to show dominant negative effects either in vivo or in vitro in the presence of the wild-type protein (33). These results corroborate our findings with the Drosophila A326T mutant.…”

Section: Discussionsupporting

confidence: 89%

“…These residues lie in predicted ␣ helices that correspond to the A and D3 helices in the linker and helicase domains, respectively, of T7 gp4. In the T7 gp4, the A helix of one protomer participates in the subunit interface with the D2 and D3 helices of the adjacent protomer (10), and amino acid substitutions near Ile 334 (Ala 359 in T7 gp4) show specific defects in translocation on single-stranded DNA and in helix unwinding (33).…”

Section: Discussionmentioning

confidence: 99%

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Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

Matsushima

Kaguni

2007

Journal of Biological Chemistry

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We report the cloning and molecular analysis of Drosophila mitochondrial DNA helicase (d-mtDNA helicase) homologous to human TWINKLE, which encodes one of the genes responsible for autosomal dominant progressive external ophthalmoplegia. An RNA interference construct was designed that reduces expression of d-mtDNA helicase to an undetectable level in Schneider cells. RNA interference knockdown of d-mtDNA helicase decreases the copy number of mitochondrial DNA (mtDNA) ϳ5-fold. In a corollary manner, overexpression of d-mtDNA helicase increases mtDNA levels 1.4-fold. Overexpression of helicase active site mutants K388A and D483A results in a severe depletion of mtDNA and a dominant negative lethal phenotype. Overexpression of mutants analogous to human autosomal dominant progressive external ophthalmoplegia mutations shows differential effects. Overexpression of I334T and A442P mutants yields a dominant negative effect as for the active site mutants. In contrast, overexpression of A326T, R341Q, and W441C mutants results in increased mtDNA copy number, as observed with wild-type overexpression. Our dominant negative analysis of d-mtDNA helicase in cultured cells provides a tractable model for understanding human autosomal dominant progressive external ophthalmoplegia mutations.One of the important functions of mitochondria is the production of ATP by the oxidative phosphorylation pathway. Animal mitochondrial DNA (mtDNA) 2 encodes 13 polypeptides involved in oxidative phosphorylation, whereas all of the factors essential for mtDNA replication are encoded in the nuclear genome (1). In general, mutations in these factors result in both loss of mtDNA integrity via base substitution mutations, duplications, and deletions and mtDNA depletion (2).Autosomal dominant progressive external ophthalmoplegia (adPEO) is a human mitochondrial disorder associated with the presence of multiple deletions of mtDNA (2-5). The disease is manifest in adulthood, typically at 20 -40 years of age; symptoms include muscle weakness, wasting, exercise intolerance, ataxia, hearing loss, cardiomyopathy, and peripheral neuropathy (2). Most of the adPEO families carry heterozygous mutations in one of three genes: ANT1 (the adenine nucleotide translocator 1), POLG (mitochondrial DNA polymerase), and TWINKLE (mtDNA helicase) (2, 6 -8).TWINKLE protein shares high homology with the bacteriophage T7 gene 4 protein (T7 gp4), which contains both helicase and primase catalytic activities located in its carboxyl-and amino-terminal halves, respectively (7). Like other DNA helicases of the DnaB family, T7 gp4 functions as a hexameric ring (9 -11). The amino acid sequence of the helicase domain of T7 gp4 is well conserved in TWINKLE, whereas that of the primase domain is not. TWINKLE exhibits 5Ј-3Ј DNA helicase activity on double-stranded DNA substrates and functions in reconstituted mtDNA replication forks in vitro (12, 13). It co-localizes with mtDNA in structures designated as mitochondrial nucleoids in vivo (7). Recently, TWINKLE was demonstrated to modu...

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

Matsushima

Kaguni

2007

Journal of Biological Chemistry

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“…Most of the biochemical assays (ssDNA filter binding, dTTP hydrolysis, DNA unwinding, strand-displacement DNA synthesis, primer synthesis, and oligomerization) used in this study have been described (9,26). All reactions were carried out in a buffer containing 40 mM Tris⅐HCl (pH 7.5), 10 mM MgCl 2, 10 mM DTT, and 50 mM potassium glutamate.…”

Section: Methodsmentioning

confidence: 99%

“…Biochemical studies have identified regions critical to oligomerization of the protein, including a linker region that connects the C-terminal helicase domain of the protein to the N-terminal primase domain (8,9). Four motifs were defined based on conserved amino acid sequences among family 4 helicases.…”

mentioning

confidence: 99%

Communication between subunits critical to DNA binding by hexameric helicase of bacteriophage T7

Lee

Qimron

Richardson

2008

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

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The DNA helicase encoded by bacteriophage T7 consists of six identical subunits that form a ring through which the DNA passes. Binding of ssDNA is a prior step to translocation and unwinding of DNA by the helicase. Arg-493 is located at a conserved structural motif within the interior cavity of the helicase and plays an important role in DNA binding. Replacement of Arg-493 with lysine or histidine reduces the ability of the helicase to bind DNA, hydrolyze dTTP, and unwind dsDNA. In contrast, replacement of Arg-493 with glutamine abolishes these activities, suggesting that positive charge at the position is essential. Based on the crystallographic structure of the helicase, Asp-468 is in the range to form a hydrogen bonding with Arg-493 on the adjacent subunit. In vivo complementation results indicate that an interaction between Asp-468 and Arg-493 is critical for a functional helicase and those residues can be swapped without losing the helicase activity. This study suggests that hydrogen bonding between Arg-493 and Asp-468 from adjacent subunits is critical for DNA binding ability of the T7 hexameric helicase.DNA replication ͉ hydrogen bonding ͉ subunit interaction ͉ residue swappping T he replication, recombination, and repair of DNA all require the unwinding of duplex DNA. Such an important transformation of dsDNA into ssDNA is carried out by a group of proteins designated as DNA helicases. Underlying the overall process of unwinding dsDNA is the ability of the protein to bind to ssDNA and use the hydrolysis of a nucleoside triphosphate to translocate unidirectionally on the DNA (1, 2). Not surprisingly, DNA helicases differ in the mechanism by which they bind to DNA, translocate on ssDNA, and unwind the duplex. On the basis of their amino acid sequences and 3D structures, DNA helicases can be divided into several families or superfamilies (2, 3).The helicase encoded by gene 4 of bacteriophage T7 is one of the most extensively characterized helicases. As a member of the DnaB-like family (family 4), the gene 4 protein forms a hexameric toroid. The oligomeric structure not only gives rise to the nucleotide bindings at the subunit interfaces but also provides a central cavity through which the ssDNA can pass. Binding sites for DNA and nucleotide span between subunits, thus allowing cooperative binding of DNA and efficient coupling of nucleotide hydrolysis to movement of the DNA strand.Electron microscopy and x-ray crystallography illustrated that the protein forms a closed ring-shaped oligomeric structure composed of six or seven subunits (4-7). Biochemical studies have identified regions critical to oligomerization of the protein, including a linker region that connects the C-terminal helicase domain of the protein to the N-terminal primase domain (8, 9). Four motifs were defined based on conserved amino acid sequences among family 4 helicases. Strictly conserved residues in motifs 1 and 2 contact the nucleotide at the nucleotide binding site located at the interface of subunits. These residues participate in h...

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“…These two functions are joined covalently by a 26-amino acid linker domain that is essential for the oligomerization of T7g4p to a hexamer, the active form of the helicase (42). Notably, two amino acids that are essential for phage growth and linker function, T7g4p Ala 257 and Asp 263 (43), are conserved in yeast Mcm10p. Temperature-sensitive mcm10 mutants are located in a region that aligns with the T7 linker domain.…”

Section: Mg2mentioning

confidence: 99%

Fission Yeast Mcm10p Contains Primase Activity

Fien

Hurwitz

2006

Journal of Biological Chemistry

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DNA replication occurs through a complex series of reactions that are mechanistically coordinated at the replication fork. Studies of model DNA replication systems indicate that origin recognition by an initiator protein permits assembly and activation of the replicative helicase at the origin. Helicases unwind duplex DNA to generate a single-strand template on which primases can initiate synthesis of RNA primers that are 4 -20 nucleotides in length and that are extended by DNA polymerases to make Okazaki fragments (1). Primases are physically coupled to the replicative DNA helicases and DNA polymerases, which translocate together through the duplex, resulting in DNA unwinding coincident with synthesis of RNA primers and their extension with deoxynucleotides.In eukaryotes, the Mcm proteins are essential replication factors that were identified as proteins required for minichromosomal maintenance in a genetic screen for mutants defective in initiation of replication (2). Six of these members are sequencerelated proteins, Mcm2-7, which interact to form a hexameric complex. Although a substantial body of data suggests that the Mcm2-7p complex acts as the replicative helicase (3), helicase activity has been detected only in the Mcm4-6-7 subcomplex (4 -6). Prior to the initiation of replication, the Mcm2-7 complex associates with the initiator at replication origins. At the G 1 /S transition, both S phase cyclin-dependent kinase and Cdc7p-Dbf4p kinase activities rise, promoting the maturation of the pre-replicative complex to the preinitiation complex (7). Phosphorylation of the chromatin-bound Mcm2-7 complex by the Cdc7-Dbf4 kinase (8) Previously, we reported that, in vitro, S. pombe Mcm10p (amino acids 1-593; SpMcm10p) binds preferentially to singlestranded DNA (ssDNA) and to the large subunit of the pol ␣-primase complex and activates its DNA synthetic activity (25). In addition, we found that Mcm10p facilitates the binding of the pol ␣-primase complex to primed DNA and forms a stable ternary complex, suggesting that Mcm10p recruits the pol ␣-primase complex to template DNA. In keeping with these in vitro findings, Mcm10p was shown recently to stabilize the large subunit of the pol ␣-primase complex in S. cerevisiae (15) as well as the chromatin association of the pol ␣-primase complex in S. pombe (26).In this study, we demonstrate that full-length SpMcm10p (amino acids 1-593) and its C-terminal fragment (amino acids 416 -593) contain primase activity that catalyzes the synthesis of oligoribonucleotides that are extended by Escherichia coli pol I. Primase activity both co-sedimented and co-eluted with these full-length and truncated proteins, although their hydro-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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The Linker Region between the Helicase and Primase Domains of the Gene 4 Protein of Bacteriophage T7

Cited by 32 publications

References 37 publications

Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

Communication between subunits critical to DNA binding by hexameric helicase of bacteriophage T7

Fission Yeast Mcm10p Contains Primase Activity

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