Identification of Escherichia coli DNA helicase I as the traI gene product of the F sex factor.

Abdel-Monem, Mahmoud M.; Taucher-Scholz, G.; Klinkert, Mo‐Quen

doi:10.1073/pnas.80.15.4659

Cited by 92 publications

(43 citation statements)

References 24 publications

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“…We speculate that whereas strand transfer in F does not require concomitant DNA synthesis, this event may require concomitant synthesis in other systems such as RP4. We envision the helicase domain on the relaxase providing the motive force required for F plasmid strand transfer, as previously suggested (Abdel-Monem et al, 1983), and the process of DNA replication providing the same force in RP4 plasmid strand transfer. However, this remains to be determined.…”

Section: The Relaxase Reactionmentioning

confidence: 73%

Nicking by transesterification: the reaction catalysed by a relaxase

Byrd¹,

Matson

1997

Molecular Microbiology

108

117

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SummaryDNA relaxases play an essential role in the initiation and termination of conjugative DNA transfer. Purification and characterization of relaxases from several plasmids has revealed the reaction mechanism: relaxases nick duplex DNA in a site-and strand-specific manner by catalysing a transesterification. The product of the reaction is a nicked double-stranded DNA molecule with a sequestered 3Ј-OH and the relaxase covalently bound to the 5Ј end of the cleaved strand via a phosphotyrosyl linkage. The relaxase-catalysed transesterification is isoenergetic and reversible; a second transesterification ligates the nicked DNA. However, the covalent nucleoprotein complex is relatively long-lived, a property that is likely to be essential for its role as an intermediate in the process of conjugative DNA transfer. Subsequent unwinding of the nicked DNA intermediate is required to produce the single strand of DNA transferred to the recipient cell. This reaction is catalysed by a DNA helicase, an activity intrinsic to the relaxase protein in some, but not all, plasmid systems. The first relaxase-catalysed transesterification is essential for initiation of conjugative strand transfer, whereas the second is presumably required for termination of the process. The relaxase, in conjunction with several auxiliary proteins, forms the relaxation complex or relaxosome first described nearly 30 years ago as being associated with conjugative and mobilizable plasmids.

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Section: The Relaxase Reactionmentioning

confidence: 73%

Nicking by transesterification: the reaction catalysed by a relaxase

Byrd¹,

Matson

1997

Molecular Microbiology

108

117

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“…The nicked plasmid is then unwound and the interrupted strand is transferred, with its 5' end first, to the F-recipient. Unwinding is most likely the effect of DNA helicase I, the product of the tral gene of the F plasmid [3]; tral is one of the five transfer genes (traMYDlZ) known to be directly involved in the DNA transfer step of the F-mediated DNA exchange reaction.…”

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confidence: 99%

Escherichia coli DNA helicase I

Benz

Müller

1990

European Journal of Biochemistry

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Gene tral of the Escherichia coli F sex factor which encodes DNA helicase I was subcloned in a / I PI,-based plasmid vector and expressed in a background of pL non-repressing cells. Neither the non-repressed pL promoter nor the production of a high level of functional helicase I are toxic. Enzyme purified from this source was studied in the electron microscope. The results show that helicase I binds cooperatively to single-stranded DNA. DNA covered with the helicase appears in fixed, negatively stained specimens as a smooth-contoured filament with a diameter of 12.5 0.4 nm and an axial periodicity of 7.0 & 0.2 nm. In unfixed specimens, discrete particles with axes of 12.7 f 0.5 nm and 7.2 0.5 nm are visible. They are consistent in size with helicase I monomers ( M r 180000) suggesting that the molecule is almost isometric, despite a frictional ratio of 1.71 calculated from its diffusion coefficient. Helicase 1 free of DNA appears as aggregates.For comparison, a truncated tral, lacking coding for the amino-terminus of the product, was cloned by fusing it to an MS2 replicase gene fragment. The chimeric gene product (named helicase I de129) retains strand-separating activity although it fails to show cooperative DNA binding behavior. Judged from the length of the helicase-1-specific sequence of this polypeptide, tral is located 1.3 kb nearer to the distal end of the F transfer operon compared to the position proposed in a previous genetic map. The revised location of tral has implications for understanding distal functions of the transfer operon.

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“…A combination of genetic and in vitro biochemical analyses have implicated these enzymes in various facets of nucleic acid metabolism. For example, helicase I participates in F factor-mediated conjugative DNA transfer (1). DnaB, PriA, and Rep proteins participate in DNA replication (23,24,34).…”

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confidence: 99%

Double helicase II (uvrD)-helicase IV (helD) deletion mutants are defective in the recombination pathways of Escherichia coli

1993

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The Escherichia coli helD (encoding helicase IV) and uvrD (encoding helicase H) genes have been deleted, independently and in combination, from the chromosome and replaced with genes encoding antibiotic resistance. Each deletion was verified by Southern blots, and the location of each deletion was confirmed by Pl-mediated transduction. Cell strains containing the single and double deletions were viable, indicating that helicases and IV are not essential for viability. Cell strains lacking helicase IV (&kelD) exhibited no increase in sensitivity to UV irradiation but were slightly more resistant to methyl methanesulfonate (MMS) than the isogenic wild-type cell strain. As expected, cell strains containing the helicase H deletion (AuvrD) were sensitive to both UV irradiation and MMS. The introduction of the helicase IV deletion into a AuvrD background had essentially no effect on the UV and MMS sensitivity of the cell strains analyzed. The double deletions, however, conferred a Rec-mutant phenotype for conjugational and transductional recombination in both recBCsbcB(C) and recBC sbcA backgrounds. The Rec-mutant phenotype was more profound in the recBC sbcB(C) background than In the recBC sbc4 background. The recombination-deficient phenotype indicates the direct involvement of helicase and/or helicase IV in the RecF pathway [recBC sbcB(C) background] and RecE pathway (recBC sbc,4 background) of recombination. The modest decrease in the recombination frequency observed in single-deletion mutants in the recBC sbcB(C) background suggests that either helicase is sufficient. In addition, helicase IV has been overexpressed in a tightly regulated system. The data suggest that even modest overexpression of helicase IV is lethal to the cell.

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Identification of Escherichia coli DNA helicase I as the traI gene product of the F sex factor.

Cited by 92 publications

References 24 publications

Nicking by transesterification: the reaction catalysed by a relaxase

Nicking by transesterification: the reaction catalysed by a relaxase

Escherichia coli DNA helicase I

Double helicase II (uvrD)-helicase IV (helD) deletion mutants are defective in the recombination pathways of Escherichia coli

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