Abstract
The Borrelia telomere resolvase, ResT, forms the unusual hairpin telomeres of the linear Borrelia replicons in a process referred to as telomere resolution. Telomere resolution is a DNA cleavage and rejoining reaction that proceeds from a replicated telomere intermediate in a reaction with mechanistic similarities to that catalyzed by type IB topoisomerases. Previous reports have implicated the hairpin-binding module, at the end of the N-terminal domain of ResT, in distorting the DNA between the scissile phosphates so as to promote DNA cleavage and hairpin formation by the catalytic domain. We report that unwinding the DNA between the scissile phosphates, prior to DNA cleavage, is a key cold-sensitive step in telomere resolution. Through the analysis of ResT mutants, rescued by substrate modifications that mimic DNA unwinding between the cleavage sites, we show that formation and/or stabilization of an underwound pre-cleavage intermediate depends upon cooperation of the hairpin-binding module and catalytic domain. The phenotype of the mutants argues that the pre-cleavage intermediate promotes strand ejection to favor the forward reaction and that subsequent hairpin capture is a reversible reaction step. These reaction features are proposed to promote hairpin formation over strand resealing while allowing reversal back to substrate of aborted reactions.
Highlights
An unusual but elegant solution to the end-replicationproblem for linear replicons is represented by hairpin telomeres, covalently closed DNA hairpins at the ends of the linear DNA
We investigated the twin issues of reaction directionality and hp telomere formation for telomere resolution catalyzed by ResT
Mimicking substrate unwinding can alleviate the coldsensitivity of telomere resolution Previous results suggest that ResT needs to distort the replicated telomere in some fashion between the scissile phosphates to allow DNA cleavage and subsequent hp formation (see Figure 1 and [23])
Summary
An unusual but elegant solution to the end-replicationproblem for linear replicons is represented by hairpin (hp) telomeres, covalently closed DNA hairpins at the ends of the linear DNA. Linear replicons with hp telomeres are found in the species of the genus Borrelia, in Agrobacterium tumefaciens and in a handful of bacteriophages that maintain a linear plasmid prophage [1,2,3,4]. The resulting daughter DNA molecules are covalently linked via the rTel junctions and must be separated by a specialized DNA breakage and reunion reaction, referred to as telomere resolution, that reforms the hp telomeres to allow for subsequent segregation [7,9,10]. The essential, specialized telomere resolvase that performs this reaction for Borrelia is known as ResT [11,12]. The characterized telomere resolvases of A. tumefaciens and the phage systems are often named ‘protelomerases’
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