Uncoupling the Chemical Steps of Telomere Resolution by ResT

Abstract

ResT is the telomere resolvase of the spirochete Borrelia burgdorferi, the causative agent of Lyme disease.ResT is an essential cellular function that processes replication intermediates to produce linear replicons terminated by covalently closed hairpin telomeres. ResT generates these hairpin telomeres in a reaction with mechanistic similarities to those catalyzed by type IB topoisomerases and tyrosine recombinases. We report here, that like most of the tyrosine recombinases, ResT requires interprotomer communication, likely in an in-line synapse, to activate reaction chemistry. Unlike the tyrosine recombinases, however, we infer that the cleavage and strand transfer reactions on the two sides of the replicated telomere occur nearly simultaneously. Nonetheless, the chemical steps of the forward and reverse reactions performed by ResT can occur in a non-concerted fashion (i.e. events on the two sides of the replicated telomere can occur independently). We propose that uncoupling of reaction completion on the two sides of the substrate is facilitated by an early commitment to hairpin formation that is imposed by the precleavage action of the hairpin binding module of the ResT active site.Spirochetes of the genus Borrelia are important human pathogens (see (1, 2)) that cause Lyme disease (3, 4) and relapsing fever (5). The Borrelia species possess intriguing genomes in that they are highly segmented with most of their replicons being linear DNAs terminated by covalently closed DNA hairpins, referred to as telomeres (see Ref. 6).The strategy employed by Borrelia burgdorferi to propagate its linear replicons has been described recently (see Refs. 7 and 8). Replication initiates from an internal origin and proceeds bidirectionally (9). Passage of the replication forks through both of the hairpin telomeres would produce an inverted repeat circular dimer replication intermediate. The resulting replicated telomeres are processed by DNA breakage and reunion events (telomere resolution) that liberate the linear daughters from the presumed circular dimer intermediate (10). The enzymatic activity that performs telomere resolution is an essential cellular function provided by ResT, the product of the BBB03 locus of cp26 (11,12). A similar replication strategy has been demonstrated for the N15 bacteriophage, the best characterized example of the bacteriophages that possess linear lysogenic genomes terminated by hairpin telomeres (13-15).Telomere resolution by ResT proceeds by a two-step transesterification involving an active site tyrosine (Tyr 335 ) that proceeds with the same polarity (a DNA-3Јphosphotyrosyl-enzyme intermediate) as that of the type IB topoisomerases and the site-specific recombinase class referred to as tyrosine recombinases (11,16). Nucleophilic attack by the active site tyrosine, on phosphodiester bonds separated by 6 bp on the opposite DNA strands in the replicated telomere, produces a transient covalent 3Ј-phosphotyrosyl-ResT intermediate. Subsequent nucleophilic attack of the phosphotyrosyl linkage b...

Preventing Broken Borrelia Telomeres

“…5). This does not occur easily on linear or relaxed DNA (25,32,34). Once this has occurred, DNA cleavage and hairpin formation rapidly follows, all within the context of the cross-axis complex (this study).…”

“…Our previous report that studied telomere resolution with substrates that blocked cleavage or hairpinning on one side of an rTel indicated that the chemical steps of telomere resolution could be uncoupled from each other (32). For the hairpin formation step of the reaction, this was particularly surprising as it implied that a hairpin telomere would be produced at the expense of also producing a deleterious ResT-capped double strand break.…”

“…The cleaved but not yet hairpinned right side of the substrate is apparent on the gel as the CP band; top strand cleavage is somewhat slower than bottom strand cleavage, but the major effect for hp2 formation is on the rate of the hairpinning step (Fig. 4) and (32). Upon immobilization of this substrate under these conditions, the yield of the hairpins is equalized, and the reaction proceeds at the slow rate of hp2 formation (compare hp2 appearance in the supernatant versus hp1 appearance in the bead fraction; Fig.…”

“…These extensions were added to eliminate steric clash and to increase the gel mobility differences of the substrate and products. The asterisks on the 5Ј ends represent 32 P end labels. The L sequence is derived from the sequence of the right telomere of lp17 and lp56.…”

Hairpin Telomere Resolvases

“…At this writing telomere resolvases have been purified from three phage and seven bacterial species: E. coli phage N15 (3), Klebsiella oxytoca phage φKO2, Yersinia enterocolitica phage PY54 (5), Agrobacterium tumefaciens (8), the Lyme spirochete Borrelia burgdorferi (6), the relapsing fever borreliae B. hermsii, B. parkeri, B. recurrentis, B. turicatae, and the avian spirochete B. anserina (7). The B. burgdorferi enzyme, ResT (Resolvase of Telomeres) has been the most extensively studied at the biochemical level (6,7,10,11,12,13,14,15,16,17,18,19,20,21,22,23) and is the primary focus of this review, with properties of the other enzymes noted (3,4,5,8,24). Structural studies of the Klebsiella phage φKO2 (25) and the Agrobacterium (26) resolvases have been reported and have shed additional light on reaction mechanisms and on differences between the resolvases from different organisms.…”