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J. Biol. Chem., Vol. 279, Issue 51, 53699-53706, December 17, 2004

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Catalytic Residues of the Telomere Resolvase ResT

A PATTERN SIMILAR TO, BUT DISTINCT FROM, TYROSINE RECOMBINASES AND TYPE IB TOPOISOMERASES^*

Jan Deneke, Alex B. Burgin, Sandra L. Wilson, and George Chaconas, Supported by a Scientist Award from the Alberta Heritage Fund for Medical Research and a Canada Research Chair in the Molecular Biology of Lyme Disease¶

From the Department of Biochemistry & Molecular Biology, and Department of Microbiology & Infectious Diseases, University of Calgary, Calgary, Alberta T2N 1N4, Canada and deCode BioStructures, Bainbridge Island, Washington 98110

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 co-valently 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.

Received for publication, August 6, 2004 , and in revised form, October 5, 2004.

^* This work was supported in part by the Canadian Institutes of Health Research, the Canada Research Chairs Program, and the Alberta Heritage Fund for Medical Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains additional text, Tables I and II, Figs. SF1 and SF2, and additional references.

^¶ To whom correspondence should be addressed. Tel.: 403-210-9692; Fax: 403-270-2772; E-mail: chaconas{at}ucalgary.ca.

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