Enzymic Unwinding of DNA

Abdel-Monem, Mahmoud M.; Dürwald, Hildegard; Hoffmann‐Berling, Hartmut

doi:10.1111/j.1432-1033.1976.tb10359.x

Cited by 202 publications

(78 citation statements)

References 16 publications

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“…Extensive studies have characterized the NTPase activity of TraI from plasmid F (97% identity between predicted proteins) (53,54). In good agreement with these reports the ATPase activity of TraI from plasmid R1 was dependent on the presence of a ssDNA effector and activated by Mg 2ϩ .…”

Section: Expression Purification and Atpase Activity Of R1 Traisupporting

confidence: 63%

“…In contrast to studies of the initiation process of replication at prokaryotic origins of replication, however, reconstitution of origin unwinding and the initiation of conjugative DNA synthesis in vitro has not been achieved. Although some helicases initiate the unwinding reaction from a nicked substrate, this activity has not been observed in vitro for TraI (54 (45) and (ii) the helicase activity when studied in isolation required a single-stranded tail 5Ј to the duplex (49) imply that localized duplex melting at nic is necessary to load the TraI helicase. In vitro reconstitution of helicase activity for enzymes that require single-stranded ends to enter the duplex is typically achieved when a short fragment or oligonucleotide targeted for displacement is annealed to a comparatively large single-stranded circular or linear phage DNA.…”

Section: Discussionmentioning

confidence: 99%

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Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Csitkovits

Zechner

2003

Journal of Biological Chemistry

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The IncF plasmid protein TraI functions during bacterial conjugation as a site-and strand-specific DNA transesterase and a highly processive 5 to 3 DNA helicase. The N-terminal DNA transesterase domain of TraI localizes the protein to nic and cleaves this site within the plasmid transfer origin. In the cell the C-terminal DNA helicase domain of TraI is essential for driving the 5 to 3 unwinding of plasmid DNA from nic to provide the strand destined for transfer. In vitro, however, purified TraI protein cannot enter and unwind nicked plasmid DNA and instead requires a 5 tail of singlestranded DNA at the duplex junction. In this study we evaluate the extent of single-stranded DNA adjacent to the duplex that is required for efficient TraI-catalyzed DNA unwinding in vitro. A series of linear partial duplex DNA substrates containing a central stretch of singlestranded DNA of defined length was created and its structure verified. We found that substrates containing >27 nucleotides of single-stranded DNA 5 to the duplex were unwound efficiently by TraI, whereas substrates containing 20 or fewer nucleotides were not. These results imply that during conjugation localized unwinding of >20 nucleotides at nic is necessary to initiate unwinding of plasmid DNA strands.Helicases are ubiquitous enzymes involved in nucleic acid metabolism (for review, see Refs. 1-4). The TraI protein of IncF plasmids (first characterized as DNA helicase I of Escherichia coli (5)) is essential for the transmission of bacterial genes during conjugation (6, 7). In that process a copy of a conjugative plasmid is transferred unidirectionally in single-stranded form from one bacterial cell to another (for reviews, see Refs. 8 and 9). The TraI protein of IncF plasmids contributes to conjugative transfer in several ways. It is a component of a nucleoprotein complex, the relaxosome, which assembles with site specificity at the plasmid origin of transfer (oriT). Relaxosomes initiate the series of DNA-processing reactions that prepare plasmid DNA for transfer to a recipient cell. They are common to all self-transmissible and mobilizable plasmids in different degrees of complexity (for reviews, see Refs. 8, 10, and 11). The simplest systems employ a plasmid-encoded DNA transesterase, or relaxase, protein that acts on a specific phosphodiester bond, nic, in the transfer origin. Cleavage at this site provides a point of origin for the directed 5Ј to 3Ј transmission of the plasmid genome to a recipient cell. Relaxase proteins are also active in relaxosomes containing auxiliary DNA-binding proteins of host or plasmid origin. IncF, IncW, IncP, and IncQ systems offer well studied examples (12-21). Among IncF plasmids factors known to stimulate nic cleavage include E. coli integration host factor and plasmid proteins . In the case of IncW plasmid R388, TrwA protein performs this role (13,14). The IncF system and the functionally analogous IncW-IncN family of DNA-mobilizing systems are (thus far) unique in that they additionally specify a DNA helicase activity esse...

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Section: Expression Purification and Atpase Activity Of R1 Traisupporting

confidence: 63%

Section: Discussionmentioning

confidence: 99%

Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Csitkovits

Zechner

2003

Journal of Biological Chemistry

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“…The product of the recB,C genes, exonuclease V, may use ATP hydrolysis to unwind duplex DNA prior to degradation of the resulting single strands (26). A 180,000 dalton protein (27) hydrolyzes ATP in the presence of single-stranded'DNA, and can promote the unwinding of duplex DNA in an ATP-dependent reaction (28). The latter protein differs from the T7 gene 4 protein in that approximately 85 protein molecules are required to unwind a 6000 nucleotide long duplex and the unwinding occurs in the absence of DNA synthesis.…”

Section: Methodsmentioning

confidence: 99%

Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydrolysis of nucleoside 5'-triphosphates.

Kolodner¹,

Richardson²

1977

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

116

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For the past several years we have been studying the replication of phage T7 DNA as a model for elucidating the molecular mechanisms by which a linear duplex DNA molecule is replicated. From in vivo and in vitro studies it is clear that two proteins, the phage T7-induced DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) and the protein encoded by gene 4 of phage T7, play a major role in replication. Genetic (2-4) and biochemical (5, 6) analyses show these two proteins to be essential for T7 DNA replication. The ¶7-induced DNA polymerase (7, 8) is composed of Escherichia coli thioredoxin and the gene 5 protein of the phage (1, 9, 10). The gene 4 protein has been purified with the aid of a complementation assay (11-13) to near homogeneity JFig. 2).Whereas neither the T7 DNA polymerase nor the gene 4 protein alone can synthesize DNA on duplex templates, the two proteins together catalyze extensive DNA synthesis (12). The reaction is specific for T7 DNA polymerase; neither T4 DNA polymerase nor E. coli DNA polymerases I, II, or III are effective (12). The reaction is stimulated by, but is not dependent on, the presence of rNTPs (12), as is replication in the in vitro system (5, 6). Although earlier studies (12, 14) with gene 4 protein suggested that the stimulation of DNA synthesis on duplex DNA resulted from a role of the gene 4 protein in initiation of DNA strands, our recent studies do not support such an interpretation. Our results (unpublished data) show that, in reaction mixtures containing the two proteins, synthesis initiates at single-strand breaks in duplex molecules with elongation of the polynucleotide chain proceeding in a 5' -3' direction, displacing the parental DNA strands. The number of replicative forks approximates the number of polymerase molecules, with polymerization proceeding at rates up to 6000 nucleotides per min per fork. At later times in the reaction the polymerase is displaced from the template by branch migration (15, 16) allowing it, along with part of the newly synthesized strand, to jump to the displaced strand and copy it in a gene 4 independent reaction. In this way T7 DNA polymerase and gene 4 protein synthesize self-complementary product molecules up to 120,000 nucleotides in length.Because the replication of duplex DNA by T7 DNA polymerase and gene 4 protein requires only 10 gene 4 protein molecules per polymerase molecule, it seems plausible that the gene 4 protein acts catalytically in conjunction with the polymerase to effect unwinding of the duplex DNA template. Inasmuch as this is likely to be an energy-requiring reaction, we looked for the hydrolysis of nucleoside 5'-triphosphates during the course of synthesis on duplex DNA. In this communication we show that the gene 4 protein is not only a single-strand-DNA-dependent nucleoside triphosphatase (NTPase; unspecific diphosphate phosphohydrolase, EC 3.6.1.15) but that hydrolysis of NTPs by gene 4 protein is coupled to DNA synthesis on duplex DNA. MAT...

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“…The TraI protein, also called helicase I, catalyzes the strand-and site-specific nicking of the F factor at oriT (16,22). Furthermore, TraI unwinds duplex DNA in an ATP-hydrolysis-dependent reaction (1)(2)(3). The precise functions of TraY, TraD, and TraM are not yet known.…”

mentioning

confidence: 99%

The cytoplasmic DNA-binding protein TraM binds to the inner membrane protein TraD in vitro

Disqué-Kochem

Dreiseikelmann

1997

J Bacteriol

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The cytoplasmic protein TraM is one of four essential gene products of the F factor which are involved in DNA transfer after mating pair formation. TraM binds to three specific sites within the oriT region. Besides regulation of its own synthesis, the precise function of TraM during conjugation is not yet known. In the present work, the affinity of TraM to TraD was studied in vitro by an overlay assay and by affinity chromatography. Whether the interaction between TraM and TraD causes a transient or permanent anchoring of the F factor to the site of transfer is discussed. A 35-kDa host membrane protein of yet unknown function also shows affinity to TraM and may be involved in this anchoring process as well.Bacterial conjugation is a process during which DNA is transferred from a donor to a recipient across the envelope of both cells. One of the best-studied conjugative plasmids is the F factor of Escherichia coli (for review, see references 7, 8, 11, and 27). After pilus synthesis and mating pair formation during the conjugation of the F factor, four additional gene products of the tra operon are directly involved in a successful DNA translocation across the envelope of the donor. These products are encoded by the genes traI, traY, traD, and traM. Of these four essential Tra proteins, TraI is the best characterized. The TraI protein, also called helicase I, catalyzes the strand-and site-specific nicking of the F factor at oriT (16,22). Furthermore, TraI unwinds duplex DNA in an ATP-hydrolysis-dependent reaction (1-3). The precise functions of TraY, TraD, and TraM are not yet known. It was recently shown in vivo that the nicking reaction of TraI is stimulated by TraY and the integration host factor (IHF) of E. coli (18). Both proteins bend DNA when bound to specific sites within the oriT region (14,15,17,19,26). Nelson and coworkers (18) propose that TraY and IHF form a nucleoprotein complex with oriT DNA which consequently can be recognized and nicked by TraI more efficiently.While TraI and TraM function in the processing of the F DNA, TraD seems to be a component of the DNA transfer apparatus. It is generally accepted that export of the singlestranded F DNA across the envelope of the donor cell proceeds through a complex pore formed by or with participation of various Tra proteins. One of these proteins is probably TraD. The TraD protein with a molecular mass of about 82 kDa is located in the inner membrane (12,20) and was shown to bind to DNA cellulose, indicating nonspecific binding to nucleic acids (20). The amino acid sequence deduced from the nucleotide sequence contains an ATP-binding motif (9) and preliminary data suggest a DNA-dependent ATPase activity of TraD (20).TraM is a cytoplasmic protein with a molecular mass of 14.5 kDa. In the past, investigations of the TraM protein have mainly been focused on its regulatory function. Maximal traM transcription requires TraY in addition to expression of the tra operon. TraM binds to three sites within the oriT region of F (6). Two sites with high affinity fo...

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Enzymic Unwinding of DNA

Cited by 202 publications

References 16 publications

Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydrolysis of nucleoside 5'-triphosphates.

The cytoplasmic DNA-binding protein TraM binds to the inner membrane protein TraD in vitro

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