TraI protein of plasmid R1 possesses two activities, a DNA transesterase and a highly processive 5-3 DNA helicase, which are essential for bacterial conjugation. Regulation of the functional domains of the enzyme is poorly understood. TraI cleaves supercoiled oriT DNA with site and strand specificity in vitro but fails to initiate unwinding from this site (nic). The helicase requires an extended region of adjacent single-stranded DNA to enter the duplex, yet interaction of purified TraI with oriT DNA alone or as an integral part of the IncF relaxosome does not melt sufficient duplex to load the helicase. This study aims to gain insights into the controlled initiation of both TraI-catalyzed activities. Linear double-stranded DNA substrates with a central region of sequence heterogeneity were used to trap defined lengths of R1 oriT sequence in unwound conformation. Concomitant reconstitution of TraI DNA transesterase and helicase activities was observed. Efficient helicase activity was measured on substrates containing 60 bases of open duplex but not on substrates containing <30 bases in open conformation. The additional presence of auxiliary DNA-binding proteins TraY and Escherichia coli integration host factor did not stimulate TraI activities on these substrates. This model system offers a novel approach to investigate factors controlling helicase loading and the directionality of DNA unwinding from nic.Conjugative DNA strand transfer is a complex process requiring highly specific protein-protein and protein-DNA interactions. Processing of the plasmid DNA is initiated when the relaxosome, a stable nucleoprotein complex, is formed at the transfer origin (oriT). This complex includes a DNA transesterase, also known as relaxase, which cleaves a specific phosphodiester bond, nic, within oriT and auxiliary DNA-binding proteins (1). After initial cleavage of the T-strand 1 (the DNA strand that is transferred to the recipient cell), the nascent terminus 3Ј to nic remains covalently bound to a catalytic tyrosine of the protein by a 5Ј phosphotyrosyl linkage, whereas the 3Ј end is released. This reaction is reversible, and in the absence of transfer, an equilibrium of cleaving and resealing is established. If transfer is initiated, T-DNA is unwound from its complementary strand and transmitted to the recipient bacterial cell in a process requiring the conjugative Type IV secretion machinery. Our current understanding of relaxosome assembly and function is derived from extensive in vitro and in vivo investigation of several conjugation systems (for reviews, see Refs. 2-4). Comparatively little detail is available for steps in the initiation process after relaxase-catalyzed strand cleavage.Duplex unwinding from nic releases the T-strand in singlestranded form and provides a substrate for translocation by the type IV secretion machinery. A recent report by Cascales and Christie (5) provides the first mechanistic insights into the initial engagement and subsequent progression of a T-DNA substrate via the Agrobacterium trans...

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...

Catalysis by radical enzymes dependent on coenzyme B12 (AdoCbl) relies on the reactive primary 5′‐deoxy‐5′adenosyl radical, which originates from reversible Co−C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 1012‐fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As revealed here, a displaced firm adenosine binding cavity in substrate‐loaded glutamate mutase (GM) causes a structural misfit for intact AdoCbl that is relieved by the homolytic Co−C bond cleavage. Strategically interacting adjacent adenosine‐ and substrate‐binding protein cavities provide a tight caged radical reaction space, controlling the entire radical path. The GM active site is perfectly structured for promoting radical catalysis, including “negative catalysis”, a paradigm for AdoCbl‐dependent mutases.

Catalysis by radical enzymes dependent on coenzyme B 12 (AdoCbl) relies on the reactive primary 5'-deoxy-5'adenosyl radical, which originates from reversible CoÀ C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 10 12 -fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As revealed here, a displaced firm adenosine binding cavity in substrateloaded glutamate mutase (GM) causes a structural misfit for intact AdoCbl that is relieved by the homolytic CoÀ C bond cleavage. Strategically interacting adjacent adenosine-and substrate-binding protein cavities provide a tight caged radical reaction space, controlling the entire radical path. The GM active site is perfectly structured for promoting radical catalysis, including "negative catalysis", a paradigm for AdoCbl-dependent mutases.