Mixing active-site components: A recipe for the unique enzymatic activity of a telomere resolvase

Abstract

The ResT protein, a telomere resolvase from Borrelia burgdorferi, processes replication intermediates into linear replicons with hairpin ends by using a catalytic mechanism similar to that for tyrosine recombinases and type IB topoisomerases. We have identified in ResT a hairpin binding region typically found in cut-and-paste transposases. We show that substitution of residues within this region results in a decreased ability of these mutants to catalyze telomere resolution. However, the mutants are capable of resolving heteroduplex DNA substrates designed to allow spontaneous destabilization and prehairpin formation. These findings support the existence of a hairpin binding region in ResT, the only known occurrence outside a transposase. The combination of transposaselike and tyrosine-recombinase-like domains found in ResT indicates the use of a composite active site and helps explain the unique breakage-and-reunion reaction observed with this protein. Comparison of the ResT sequence with other known telomere resolvases suggests that a hairpin binding motif is a common feature in this class of enzyme; the sequence motif also appears in the RAG recombinases. Finally, our data support a mechanism of action whereby ResT induces prehairpin formation before the DNA cleavage step.

Uncoupling the Chemical Steps of Telomere Resolution by ResT

“…We propose that an early, precleavage commitment to hairpin formation in the forward reaction explains this unusual mode of action. ResT has a composite active site; besides employing topo IB or tyrosine recombinase-like catalytic residues (16), the ResT active site also contains a hairpin binding module that acts before DNA cleavage to distort the DNA between the cleavage sites to activate cleavage and to aid in hairpin formation (21). Heteroduplexing the DNA between the cleavage sites rescues the defect of mutants in this module (21).…”

“…The action of the hairpin binding module to distort the DNA between the cleavage sites is required to activate DNA cleavage (21) and the two active site components explain the unique activity of telomere resolvases. To gain a further understanding of the mechanistic properties of this unique class of enzymes, we performed experiments to ask whether there was a requirement for concerted DNA cleavage and strand transfer for the telomere resolvase ResT.…”

Requirements for DNA hairpin formation by RAG1/2

“…The region around RAG1 W893 has been suggested to share the Tn5/Tn10/ResT hairpin-binding motif (18,30); indeed, mutations in surrounding residues are deficient in hairpinning (39).…”

“…These biochemical experiments support the notion that C1b lies extrahelically during cleavage and interacts with RAG1 and that contacts with C2t and S2b are also occurring, as suggested by our abasic screen. (30). Although W319 of Hermes has been implicated in hairpin formation (22) and appears to follow the first part of the motif, other members of the hAT transposase family do not conform to either part of this motif (37) (SI Fig.…”

“…Because W893 has been suggested to be the equivalent Trp to W298 of Tn5 (18,30), we examined the effect of the W893A mutation on hairpin formation more closely. In Mn 2ϩ , only a minor decrease in conversion of nicks to hairpins was seen with normal substrates (a reduction of one-third).…”