ResTs Relationship to Topoisomerases and Site Specific Recombinases

Topoisomerases, site-specific recombinases and telomere resolvases share a common chemical mechanism; however, the gross nature of their reactions differ because the number of DNA strands cleaved and the identity of the strands they are joined to differ for each class of enzyme. Topoisomerases cleave one (types I and III) or two strands (types II and IV) which are rejoined after rotation or strand passage to change the levels of cellular DNA supercoiling or catenation (Champoux 2001; Shuman 1998; Wang 1996). Site-specific recombinases (both serine and tyrosine classes) cleave four DNA strands and join them to new partners to produce recombinant products. Telomere resolution is a unique reaction whose complexity lies midway in this continuum. Here, two DNA strands are cleaved and rejoined to the opposing complementary strand. The fact that strand transfer occurs to a new partner strand makes the resolution reaction somewhat akin to a recombination reaction in which strand transfer occurs within the rTel substrate rather than to a recombination partner duplex (Kobryn et al. 2005). These differences in the global nature of the reactions catalysed are accompanied by different requirements for the coordination of active sites and for the timing of the cleavage and strand joining steps on each strand of the target site.

It was, therefore, of interest to investigate these issues for both the telomere resolution and fusion reactions in order to determine the depth of the apparent relationship of ResT to the type IB topoisomerases, on the one hand, and to the tyrosine recombinases on the other. In contrast to the type IB topoiso-merases which act as monomers, ResT is not active on isolated half-sites or hp telomeres. ResT-ResT communication through, at the very least, an in-line synapse of such sites mimicking the structure of the rTel substrate seems to be required to activate DNA cleavage (Kobryn et al. 2005).

Tyrosine recombinases catalyse reactions in which two sites are brought together followed by execution of pairwise DNA cleavage and strand transfer reactions of the equivalent strand in each duplex to form a Holliday junction intermediate. The junction is then isomerized to activate the remaining pair of active sites for cleavage and strand transfer of the other pair of strands to produce the recombinant product (Van Duyne 2002). In contrast to this, ResT is inferred to catalyse nearly simultaneous cleavage and strand transfer reactions on the two halves of its substrate. Despite this, for both steps in both the forward and reverse reactions, the chemical steps can be uncoupled from each other. This unusual combination of characteristics is consistent with the pre-cleavage action of the hairpin-binding module of ResT imposing a commitment to completion of the cleavage and strand transfer cycle (Kobryn et al. 2005).

The pattern of active site residues and the reaction characteristics just described above are suggestive of a situation in which part of an ancestral cut-

and-paste transposase was fused to part of an ancestral tyrosine recombinase resulting in this new activity of telomere resolution. Conversion or co-option of either a plasmid/chromosome dimer resolving enzyme (i.e. XerC/(D)) or a bacteriophage integrase (e.g. \ integrase) in this manner in a bacterium with a circular genome could result in the genome's linearization and fragmentation. Discovery of further ancestral properties in ResT and its reactions from either partner in the proposed fusion would lend further support to this hypothesis.

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