Enzymatic unwinding of DNA

Abdel-Monem, Mahmoud M.; Lauppe, Hans-Frieder; Kartenbeck, Jürgen; Dürwald, Hildegard; Hoffmann‐Berling, Hartmut

doi:10.1016/s0022-2836(77)80083-1

Cited by 69 publications

(19 citation statements)

References 22 publications

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“…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%

“…Preparation and Verification of the Helicase Substrate Structures-The TraI protein of plasmid F is known to require a region of ssDNA adjacent to the duplex to facilitate its DNA unwinding activity (47)(48)(49). To evaluate the requirement of this enzyme for a 5Ј ssDNA tail, we chose to create a series of linear partial duplex substrates containing a centrally located single-stranded gap of variable length.…”

Section: Expression Purification and Atpase Activity Of R1 Traimentioning

confidence: 99%

“…Reconstitution of the duplex-unwinding activity in vitro requires a region of ssDNA adjacent to the duplex (49). In early work Hoffmann-Berling and co-workers (47) estimated that the length of ssDNA required by TraI on the duplex 5Ј end lies within an approximate range of 12-200 nt.…”

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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: Discussionmentioning

confidence: 99%

Section: Expression Purification and Atpase Activity Of R1 Traimentioning

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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“…de Recondo, personal communication). This activity from prokaryotes is also excluded from DNA-cellulose [21,22] or from DNAagarose [23,24] at a high salt concentration. More work is needed in order to verify these hypotheses and identify the possible regulatory factor/mechanism of DNA polymerase enzyme.…”

Section: Discussionmentioning

confidence: 99%

In vitro activation of DNA polymerase‐α by a protein kinase in chick embryo

1981

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“…The rep helicase separates the strands in the direction of DNA replication on the leading strand. A general method to assay the action of DNA helicases is the degradation of single-stranded DNA generated by SI-endonuclease [6]. Partially unwound duplex DNA cannot be assayed by this method, as the strands will renature before completion of endonucleolytic degradation or endonucleolytic digest of partial duplexes cannot be distinguished from degradation of completely separated strands.…”

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Functional aspects of Escherichia coli rep helicase in unwinding and replication of DNA

Bäumel

Meyer

Geider

1984

European Journal of Biochemistry

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The gene for Escherichia coli rep helicase (rep protein) was subcloned in a pBR plasmid and the protein overproduced in cells transformed with the hybrid DNA. The effect of purified enzyme on strand unwinding and DNA replication was investigated by electron microscopy. The templates used were partial duplexes of viral DNA from bacteriophage fd: :Tn5 and reannealed DNA from bacteriophage Mu. The experiments with the two DNA species show DNA unwinding uncoupled from replication. The single-stranded phage fd: :Tn5 DNA with the inverted repeat of transposon Tn5 could be completely replicated in the presence of the E. coli enzymes rep helicase, DNA binding protein I, RNA polymerase and DNA polymerase I11 holoenzyme. A block in the unwinding step increases secondary initiation events in single-stranded parts of the template, as DNA polymerase I11 holoenzyme cannot switch across the stem structure of the transposon.The Escherichia coli gene rep has been implicated with replication of small bacteriophages [l]. The enzymatic involvement of rep helicase in strand unwinding has been shown in vitro for replication of double-stranded phage 4x174 [2] and phage fd DNA [3].The action of rep helicase in the fork depends on ATP or dATP as energy source [4]. The polarity of unwinding by rep helicase is opposite to the direction of strand separation found for other E. coli DNA helicases [5]. The rep helicase separates the strands in the direction of DNA replication on the leading strand. A general method to assay the action of DNA helicases is the degradation of single-stranded DNA generated by SI-endonuclease [6]. Partially unwound duplex DNA cannot be assayed by this method, as the strands will renature before completion of endonucleolytic degradation or endonucleolytic digest of partial duplexes cannot be distinguished from degradation of completely separated strands. However, electron microscopy is a convenient tool to study partial denaturation of double-stranded DNA [7].The E. coli DNA helicases I, I1 and 111 require a DNA duplex to be partially single-stranded in order to unwind DNA [8]. E. coli rep helicase can initiate DNA unwinding at a nick if a helper protein like gene 2 protein for phage fd [3] or gene A protein for phage 4x174 DNA is present [9]. In the absence of these phage-specific proteins, unwinding by rep helicase also depends on single-stranded regions in the duplex DNA [lo]. The enzyme might also be involved in replication of the host chromosome. Defects in the coding gene results in increased fork initiation [ll]. The rep mutation does not seem compatible with the non-functional uvrD gene product (DNA helicase 11) [12]. It has therefore been assumed that E. coli rep helicase and DNA helicase I1 simultaneously unwind the two strands in a replication fork.Here we will describe electron microscopical investigations on enzymatic strand separation by E. coli rep helicase in the presence of E. coli DNA binding protein I. It will be shown that DNA replication by DNA polymerase I11 holoenzyme on a Abbreviation. RF, r...

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Enzymatic unwinding of DNA

Cited by 69 publications

References 22 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

In vitro activation of DNA polymerase‐α by a protein kinase in chick embryo

Functional aspects of Escherichia coli rep helicase in unwinding and replication of DNA

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