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Branch migration enzyme as a Brownian ratchet.

ABSTRACT: In recent years, it has been shown that helicases are able to perform functions beyond their traditional role in unwinding of double-stranded nucleic acids; yet the mechanistic aspects of these different activities are not clear. Our kinetic studies of Holliday junction branch migration catalysed by a ring-shaped helicase, T7 gp4, show that heterology of as little as a single base stalls catalysed branch migration. Using single-molecule analysis, one can locate the stall position to within a few base pairs of the heterology. Our data indicate that the presence of helicase alone promotes junction unfolding, which accelerates spontaneous branch migration, and individual time traces reveal complex trajectories consistent with random excursions of the branch point. Our results suggest that instead of actively unwinding base pairs as previously thought, the helicase exploits the spontaneous random walk of the junction and acts as a Brownian ratchet, which walks along duplex DNA while facilitating and biasing branch migration in a specific direction.

SUBMITTER: Rasnik I

PROVIDER: S-EPMC2435128 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Branch migration enzyme as a Brownian ratchet.

Rasnik Ivan I Jeong Yong-Joo YJ McKinney Sean A SA Rajagopal Vaishnavi V Patel Smita S SS Ha Taekjip T

The EMBO journal 20080529 12

In recent years, it has been shown that helicases are able to perform functions beyond their traditional role in unwinding of double-stranded nucleic acids; yet the mechanistic aspects of these different activities are not clear. Our kinetic studies of Holliday junction branch migration catalysed by a ring-shaped helicase, T7 gp4, show that heterology of as little as a single base stalls catalysed branch migration. Using single-molecule analysis, one can locate the stall position to within a few ...[more]

PMID: 18511910

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Project description:Bacterial plasmids are extrachromosomal DNA that provides selective advantages for bacterial survival. Plasmid partitioning can be remarkably robust. For high-copy-number plasmids, diffusion ensures that both daughter cells inherit plasmids after cell division. In contrast, most low-copy-number plasmids need to be actively partitioned by a conserved tripartite ParA-type system. ParA is an ATPase that binds to chromosomal DNA; ParB is the stimulator of the ParA ATPase and specifically binds to the plasmid at a centromere-like site, parS. ParB stimulation of the ParA ATPase releases ParA from the bacterial chromosome, after which it takes a long time to reset its DNA-binding affinity. We previously demonstrated in vitro that the ParA system can exploit this biochemical asymmetry for directed cargo transport. Multiple ParA-ParB bonds can bridge a parS-coated cargo to a DNA carpet, and they can work collectively as a Brownian ratchet that directs persistent cargo movement with a ParA-depletion zone trailing behind. By extending this model, we suggest that a similar Brownian ratchet mechanism recapitulates the full range of actively segregated plasmid motilities observed in vivo. We demonstrate that plasmid motility is tuned as the replenishment rate of the ParA-depletion zone progressively increases relative to the cargo speed, evolving from diffusion to pole-to-pole oscillation, local excursions, and, finally, immobility. When the plasmid replicates, the daughters largely display motilities similar to that of their mother, except that when the single-focus progenitor is locally excursive, the daughter foci undergo directed segregation. We show that directed segregation maximizes the fidelity of plasmid partition. Given that local excursion and directed segregation are the most commonly observed modes of plasmid motility in vivo, we suggest that the operation of the ParA-type partition system has been shaped by evolution for high fidelity of plasmid segregation.

Dataset Information

Branch migration enzyme as a Brownian ratchet.

Publications

Branch migration enzyme as a Brownian ratchet.

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