Functional Domains in Protein TrwC of Plasmid R388: Dissected DNA Strand Transferase and DNA Helicase Activities Reconstitute Protein Function

Llosa, Matxalen; Grandoso, Guadalupe; Hernando, Miguel; Cruz, Fernando de la

doi:10.1006/jmbi.1996.0623

Cited by 68 publications

(94 citation statements)

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“…Recruitment of replicative helicases to replication origins typically requires the activity of sequencespecific DNA-binding proteins that confer site specificity to the helicase activity as well as induce localized distortions in the duplex to provide regions of ssDNA for the helicase to bind (46). The IncF-IncW family of DNA/mobilizing systems achieve site specificity in oriT DNA unwinding by physical linkage of the conjugative helicase to an N-terminal DNA relaxase domain (34,38,55,56), 2 which acts to cleave the transfer origin with nt precision, and presumably via interactions with additional components of the relaxosome. 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.…”

Section: Discussionmentioning

confidence: 99%

“…The DNA helicase activities are located in C-terminal domains of the bifunctional F TraI and R388 TrwC proteins (28 -33). For reasons that are poorly understood, efficient gene transfer requires physical linkage of the relaxase and helicase domains (7,34).This bifunctional arrangement of DNA transesterase and helicase activities is shared by adeno-associated virus replication initiation (Rep) 1 proteins (35). As revealed by very recent structural data, the endonuclease domain of adeno-associated virus-5 Rep proteins (36) bear the closest structural and functional relatedness to the N-terminal DNA transesterase domains of the F TraI and R388 TrwC proteins (37).…”

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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

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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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“…TrwC, the relaxase of plasmid R388, is a bifunctional protein with an N-terminal relaxase domain and a C-terminal DNA helicase domain (15). The atomic structures of the relaxase domains of TrwC and TraI of plasmid F have been solved (16,17).…”

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Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells

Draper

César

Machón

et al. 2005

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

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Conjugative relaxases are the proteins that initiate bacterial conjugation by a site-specific cleavage of the transferred DNA strand. In vitro, they show strand-transferase activity on single-stranded DNA, which suggests they may also be responsible for recircularization of the transferred DNA. In this work, we show that TrwC, the relaxase of plasmid R388, is fully functional in the recipient cell, as shown by complementation of an R388 trwC mutant in the recipient. TrwC transport to the recipient is also observed in the absence of DNA transfer, although it still requires the conjugative coupling protein. In addition to its role in conjugation, TrwC is able to catalyze site-specific recombination between two origin of transfer (oriT) copies. Mutations that abolish TrwC DNA strandtransferase activity also abolish oriT-specific recombination. A plasmid containing two oriT copies resident in the recipient cell undergoes recombination when a TrwC-piloted DNA is conjugatively transferred into it. Finally, we show TrwC-dependent integration of the transferred DNA into a resident oriT copy in the recipient cell. Our results indicate that a conjugative relaxase is active once in the recipient cell, where it performs the nicking and strand-transfer reactions that would be required to recircularize the transferred DNA. This TrwC site-specific integration activity in recipient cells may lead to future biotechnological applications.bacterial conjugation ͉ plasmid R388 ͉ site-specific integration ͉ strand transferase ͉ TrwC B acterial conjugation is a widespread mechanism for horizontal DNA transfer among prokaryotes. Under laboratory conditions, conjugation has also been reported between bacteria and eukaryotic cells (1-3). Any DNA molecule containing a short segment called the origin of transfer (oriT) can be conjugatively transferred to a recipient cell if the rest of the conjugation machinery is provided, either in cis or in trans. The transfer apparatus can be divided into three functional modules (4 -6). (i) A nucleoprotein complex known as relaxosome consists of oriT-binding proteins and their cognate DNA. These proteins include one relaxase plus one or more accessory nicking proteins; the relaxase introduces a site-specific nick at the oriT and remains covalently bound to the 5Ј end of the strand that is to be transferred. (ii) A type IV secretion system (T4SS) is a multiprotein complex spanning the inner and outer membranes through which the substrate is secreted. (iii) The conjugative coupling protein (T4CP) is responsible for connecting the two other functional modules. Current models for DNA transfer through T4SS, in both conjugation and the Agrobacterium tumefaciens T-DNA transfer system (7,8), postulate that a T4CP recruits the relaxosome to the T4SS via protein-protein interactions.For Ͼ20 years, models for conjugative DNA transport have postulated that the relaxase catalyzes the final recircularization step of the transferred DNA because of its strand-transfer activity (8, 9). Nevertheless, it was not until r...

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“…Relaxases cleave ssDNA in a Mg 2ϩ -dependent transesterification reaction that proceeds through a stable phosphotyrosyl intermediate (9)(10)(11)(12)(13)(14)(15). At the end of conjugative transfer, relaxases are thought to ligate the plasmid ends together (9,(16)(17)(18)(19).…”

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Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100

Harley

Schildbach

2003

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

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Conjugative plasmid transfer is an important mechanism for diversifying prokaryotic genomes and disseminating antibiotic resistance. Relaxases are conjugative plasmid-encoded proteins essential for plasmid transfer. Relaxases bind and cleave one plasmid strand siteand sequence-specifically before transfer of the cleaved strand. TraI36, a domain of F plasmid TraI that contains relaxase activity, binds a plasmid sequence in single-stranded form with subnanomolar KD and high sequence specificity. Despite 91% amino acid sequence identity, TraI36 domains from plasmids F and R100 discriminate between binding sites. The binding sites differ by 2 of 11 bases, but both proteins bind their cognate site with three orders of magnitude higher affinity than the other site. To identify specificity determinants, we generated variants having R100 amino acids in the F TraI36 background. Although most retain F specificity, the Q193R͞R201Q variant binds the R100 site with 10-fold greater affinity than the F site. The reverse switch (R193Q͞Q201R) in R100 TraI36 confers a wild-type F specificity on the variant. Nonadditivity of individual amino acid and base contributions to recognition suggests that the specificity difference derives from multiple interactions. The F TraI36 crystal structure shows positions 193 and 201 form opposite sides of a pocket within the binding cleft, suggesting binding involves knob-into-hole interactions. Specificity is presumably modulated by altering the composition of the pocket. Our results demonstrate that F-like relaxases can switch between highly sequence-specific recognition of different sequences with minimal amino acid substitution.

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Functional Domains in Protein TrwC of Plasmid R388: Dissected DNA Strand Transferase and DNA Helicase Activities Reconstitute Protein Function

Cited by 68 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

Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells

Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100

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