Catalytic Residues of the Telomere Resolvase ResT

Abstract

ResT is a member of the telomere resolvases, a newly discovered class of DNA breakage and reunion enzymes. These enzymes are involved in the formation of covalently closed hairpin DNA ends that are found in linear prokaryotic chromosomes and plasmids. The hairpins are generated by telomere resolution, where the replicated linear DNA ends are processed by DNA breakage followed by joining of DNA free ends to the complementary strand of the same molecule. Previous studies have shown that ResT catalyzes hairpin formation through a two-step transesterification similar to tyrosine recombinases and type IB topoisomerases. In the present study we have probed the reaction mechanism of ResT. The enzyme was found to efficiently utilize a substrate with a 5-bridging phosphorothiolate at each cleavage site, similar to tyrosine recombinases/type IB topoisomerases. Using such a substrate to trap the covalent protein-DNA intermediate, coupled with affinity purification and mass spectroscopy, we report a new, non-radioactive approach to directly determine the position of the amino acid in the protein, which is linked to the DNA. We report that tyrosine 335 is the active site nucleophile in ResT, strengthening the link between ResT and tyrosine recombinases/type IB topoisomerases. However, a distinct pattern of catalytic residues with similarities, but distinct differences from the above enzymes was suggested. The differences include the apparent absence of a general acid catalyst, as well as the dispensability of the final histidine in the RKHRHY hexad. Finally, two signature motifs (GRR(2X)E(6X)F and LGH(4 -6X)T(3X)Y) near the catalytic residues of aligned telomere resolvases are noted.Telomere resolvases are a unique family of recently discovered DNA breakage and reunion enzymes (Refs. 1, 2, and see Refs. 3 and 4 for recent reviews). Telomere resolvases promote the formation of covalently closed hairpin ends or "telomeres" on DNA through a two-step transesterification (2, 5); they are required for the replication of linear DNA molecules with hairpin ends found in several phage and bacterial species (3, 4, 6 -8).Only four enzymes of this class have been identified by biochemical analysis: the phage encoded TelN of N15 (1), the bacterial protein ResT of Borrelia burgdorferi (2), Tel PY54 of the Yersinia enterocolitica phage PY54 (9), and TelK of the Klebsiella phage KO2 (10). ResT, which is the subject of this study, is essential for B. burgdorferi (11). This bacterium is the causative agent of Lyme disease and has a segmented genome that includes a linear chromosome and 12 linear extrachromosomal elements, all terminated by covalently closed hairpin ends (12, 13). Replication of these linear molecules is believed to occur through bidirectional replication from a centrally located origin (14) followed by telomere resolution (15) mediated by ResT (2, 16), as shown in Fig. 1. Telomere resolution involves breakage of two phosphodiester bonds in a doublestranded DNA substrate (one in each strand) and joining of each end with the opp...

Uncoupling the Chemical Steps of Telomere Resolution by ResT

“…The forward and reverse reactions performed by ResT. A, in telomere resolution by ResT, a replicated telomere is resolved into two covalently closed hairpin ends through a two-step transesterification (11,16).…”

“…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.…”

“…The composite active site of ResT has catalytic residues similar to those described for type IB topoisomerases or tyrosine recombinases (16) but also incorporates a hairpin binding module similar to that found in cut-and-paste transposases of the Tn5 and Tn10 elements, which use a DNA hairpin intermediate (18 -20). 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.…”

“…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.…”

Construction and Characterization of a Borrelia burgdorferi Strain with Conditional Expression of the Essential Telomere Resolvase, ResT

“…ResT has been extensively characterized, and its mechanism is well defined (12,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32). The enzyme is mechanistically similar to type IB topoisomerases and tyrosine recombinases.…”