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Identification of the Determinant Conferring Permissive Substrate Usage in the Telomere Resolvase, ResT

2009; Elsevier BV; Volume: 284; Issue: 35 Linguagem: Inglês

10.1074/jbc.m109.023549

ISSN

1083-351X

Autores

Tara J. Moriarty, George Chaconas,

Tópico(s)

Toxoplasma gondii Research Studies

Resumo

Linear genome stability requires specialized telomere replication and protection mechanisms. A common solution to this problem in non-eukaryotes is the formation of hairpin telomeres by telomere resolvases (also known as protelomerases). These enzymes perform a two-step transesterification on replication intermediates to generate hairpin telomeres using an active site similar to that of tyrosine recombinases and type IB topoisomerases. Unlike phage telomere resolvases, the telomere resolvase from the Lyme disease pathogen Borrelia burgdorferi (ResT) is a permissive enzyme that resolves several types of telomere in vitro. However, the ResT region and residues mediating permissive substrate usage have not been identified. The relapsing fever Borrelia hermsii ResT exhibits a more restricted substrate usage pattern than B. burgdorferi ResT and cannot efficiently resolve a Type 2 telomere. In this study, we determined that all relapsing fever ResTs process Type 2 telomeres inefficiently. Using a library of chimeric and mutant B. hermsii/B. burgdorferi ResTs, we mapped the determinants in B. burgdorferi ResT conferring the ability to resolve multiple Type 2 telomeres. Type 2 telomere resolution was dependent on a single proline in the ResT catalytic region that was conserved in all Lyme disease but not relapsing fever ResTs and that is part of a 2-amino acid insertion absent from phage telomere resolvase sequences. The identification of a permissive substrate usage determinant explains the ability of B. burgdorferi ResT to process the 19 unique telomeres found in its segmented genome and will aid further studies on the structure and function of this essential enzyme. Linear genome stability requires specialized telomere replication and protection mechanisms. A common solution to this problem in non-eukaryotes is the formation of hairpin telomeres by telomere resolvases (also known as protelomerases). These enzymes perform a two-step transesterification on replication intermediates to generate hairpin telomeres using an active site similar to that of tyrosine recombinases and type IB topoisomerases. Unlike phage telomere resolvases, the telomere resolvase from the Lyme disease pathogen Borrelia burgdorferi (ResT) is a permissive enzyme that resolves several types of telomere in vitro. However, the ResT region and residues mediating permissive substrate usage have not been identified. The relapsing fever Borrelia hermsii ResT exhibits a more restricted substrate usage pattern than B. burgdorferi ResT and cannot efficiently resolve a Type 2 telomere. In this study, we determined that all relapsing fever ResTs process Type 2 telomeres inefficiently. Using a library of chimeric and mutant B. hermsii/B. burgdorferi ResTs, we mapped the determinants in B. burgdorferi ResT conferring the ability to resolve multiple Type 2 telomeres. Type 2 telomere resolution was dependent on a single proline in the ResT catalytic region that was conserved in all Lyme disease but not relapsing fever ResTs and that is part of a 2-amino acid insertion absent from phage telomere resolvase sequences. The identification of a permissive substrate usage determinant explains the ability of B. burgdorferi ResT to process the 19 unique telomeres found in its segmented genome and will aid further studies on the structure and function of this essential enzyme. Replication and protection of telomeric DNA are required to ensure the genomic stability of all organisms with linear replicons. Until quite recently, it was assumed that linearity is a property confined to the replicons of eukaryotes and certain primarily eukaryotic viruses. However, a growing body of evidence indicates that linear DNA is also found in a broad range of bacteriophages (1.Mellado R.P. Peñalva M.A. Inciarte M.R. Salas M. 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Cell. 1982; 28: 315-324Abstract Full Text PDF PubMed Scopus (193) Google Scholar, 14.González A. Talavera A. Almendral J.M. Viñuela E. Nucleic Acids Res. 1986; 14: 6835-6844Crossref PubMed Scopus (101) Google Scholar, 15.Cotmore S.F. Tattersall P. Adv. Virus Res. 1987; 33: 91-174Crossref PubMed Scopus (385) Google Scholar, 16.Delaroque N. Müller D.G. Bothe G. Pohl T. Knippers R. Boland W. Virology. 2001; 287: 112-132Crossref PubMed Scopus (98) Google Scholar). Hairpin DNA is not recognized as a double-strand break, and continuous synthesis of DNA around the hairpin loop abolishes the end replication problem. However, mother and daughter replicons are covalently linked at the junction of their telomeres following DNA replication; separation of the two replicons and formation of new hairpin telomeres require a DNA breakage and reunion process referred to as telomere resolution (17.Chaconas G. Stewart P.E. Tilly K. Bono J.L. Rosa P. EMBO J. 2001; 20: 3229-3237Crossref PubMed Scopus (76) Google Scholar, 18.Chaconas G. Mol. Microbiol. 2005; 58: 625-635Crossref PubMed Scopus (50) Google Scholar). Resolution of the linear chromosome and plasmids in Borrelia species and of the linear plasmid prophages from Escherichia coli, Yersinia enterocolitica, and Klebsiella oxytoca is performed by telomere resolvases (also referred to as protelomerases) (5.Hertwig S. Klein I. Lurz R. Lanka E. Appel B. Mol. Microbiol. 2003; 48: 989-1003Crossref PubMed Scopus (64) Google Scholar, 19.Kobryn K. Chaconas G. Mol. Cell. 2002; 9: 195-201Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 20.Deneke J. Ziegelin G. Lurz R. Lanka E. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7721-7726Crossref PubMed Scopus (74) Google Scholar, 21.Huang W.M. Joss L. Hsieh T. Casjens S. J. Mol. Biol. 2004; 337: 77-92Crossref PubMed Scopus (30) Google Scholar). A growing number of candidate telomere resolvases have been identified in the genomes of eukaryotic viruses, phages, and bacteria (22.Deneke J. Burgin A.B. Wilson S.L. Chaconas G. J. Biol. Chem. 2004; 279: 53699-53706Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 23.Oakey H.J. Cullen B.R. Owens L. J. Appl. Microbiol. 2002; 93: 1089-1098Crossref PubMed Scopus (75) Google Scholar). Telomere resolvases are DNA cleavage and rejoining enzymes related to tyrosine recombinases and type 1B topoisomerases (19.Kobryn K. Chaconas G. Mol. Cell. 2002; 9: 195-201Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 21.Huang W.M. Joss L. Hsieh T. Casjens S. J. Mol. Biol. 2004; 337: 77-92Crossref PubMed Scopus (30) Google Scholar, 22.Deneke J. Burgin A.B. Wilson S.L. Chaconas G. J. Biol. Chem. 2004; 279: 53699-53706Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 24.Deneke J. Ziegelin G. Lurz R. Lanka E. J. Biol. Chem. 2002; 277: 10410-10419Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 25.Aihara H. Huang W.M. Ellenberger T. Mol. Cell. 2007; 27: 901-913Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Telomere resolvase catalyzes a two-step transesterification reaction in which staggered cuts are introduced 6 bp apart on either side of the axis of symmetry in the replicated telomere substrate (5.Hertwig S. Klein I. Lurz R. Lanka E. Appel B. Mol. Microbiol. 2003; 48: 989-1003Crossref PubMed Scopus (64) Google Scholar, 19.Kobryn K. Chaconas G. Mol. Cell. 2002; 9: 195-201Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 21.Huang W.M. Joss L. Hsieh T. Casjens S. J. Mol. Biol. 2004; 337: 77-92Crossref PubMed Scopus (30) Google Scholar, 24.Deneke J. Ziegelin G. Lurz R. Lanka E. J. Biol. Chem. 2002; 277: 10410-10419Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Cleavage is accompanied by the formation of a 3′-phosphotyrosyl protein-DNA linkage. Subsequent nucleophilic attack on opposing strands by the free 5′-OH groups in the nicked substrate creates covalently closed hairpin telomeres. A recent crystal structure of the Klebsiella phage telomere resolvase (TelK) in complex with its substrate identified the residues involved in catalysis (25.Aihara H. Huang W.M. Ellenberger T. Mol. Cell. 2007; 27: 901-913Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar); all but one of these residues are conserved in all telomere resolvases (22.Deneke J. Burgin A.B. Wilson S.L. Chaconas G. J. Biol. Chem. 2004; 279: 53699-53706Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), implying that the basic catalytic mechanism underlying telomere resolution is conserved. However, telomere resolvase sequences vary substantially outside of the central catalytic region (25.Aihara H. Huang W.M. Ellenberger T. Mol. Cell. 2007; 27: 901-913Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 26.Tourand Y. Lee L. Chaconas G. Mol. Microbiol. 2007; 64: 580-590Crossref PubMed Scopus (19) Google Scholar), and the enzymes characterized to date demonstrate important differences in substrate usage that likely reflect functionally distinct mechanisms of substrate interaction. The Borrelia burgdorferi telomere resolvase, ResT, appears to be particularly divergent. It is substantially smaller than phage telomere resolvases, and unlike its phage counterparts (5.Hertwig S. Klein I. Lurz R. Lanka E. Appel B. Mol. Microbiol. 2003; 48: 989-1003Crossref PubMed Scopus (64) Google Scholar, 20.Deneke J. Ziegelin G. Lurz R. Lanka E. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7721-7726Crossref PubMed Scopus (74) Google Scholar, 21.Huang W.M. Joss L. Hsieh T. Casjens S. J. Mol. Biol. 2004; 337: 77-92Crossref PubMed Scopus (30) Google Scholar), it cannot efficiently resolve negatively supercoiled DNA (19.Kobryn K. Chaconas G. Mol. Cell. 2002; 9: 195-201Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 27.Bankhead T. Kobryn K. Chaconas G. Mol. Microbiol. 2006; 62: 895-905Crossref PubMed Scopus (24) Google Scholar), presumably reflecting differences in the substrates resolved by phage and Borrelia telomere resolvases in vivo. On the other hand, B. burgdorferi ResT can fuse hairpin telomeres in a reversal of the resolution reaction (28.Kobryn K. Chaconas G. Mol. Cell. 2005; 17: 783-791Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), a function that is not shared with the phage telomere resolvase TelK (25.Aihara H. Huang W.M. Ellenberger T. Mol. Cell. 2007; 27: 901-913Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). It can also synapse replicated telomeres and catalyze the formation of Holliday junctions (29.Kobryn K. Briffotaux J. Karpov V. Mol. Microbiol. 2009; 71: 1117-1130Crossref PubMed Scopus (13) Google Scholar). The ability of ResT to promote hairpin fusion has been proposed as the mechanism underlying the ongoing genetic rearrangements that are a prominent feature of the B. burgdorferi genome (18.Chaconas G. Mol. Microbiol. 2005; 58: 625-635Crossref PubMed Scopus (50) Google Scholar, 28.Kobryn K. Chaconas G. Mol. Cell. 2005; 17: 783-791Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Finally, B. burgdorferi ResT can tolerate a surprising amount of variation in its substrate (30.Tourand Y. Kobryn K. Chaconas G. Mol. Microbiol. 2003; 48: 901-911Crossref PubMed Scopus (35) Google Scholar, 31.Tourand Y. Deneke J. Moriarty T.J. Chaconas G. J. Biol. Chem. 2009; 284: 7264-7272Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), a feature that is not shared by phage telomere resolvases (21.Huang W.M. Joss L. Hsieh T. Casjens S. J. Mol. Biol. 2004; 337: 77-92Crossref PubMed Scopus (30) Google Scholar). Although B. burgdorferi ResT appears to be more permissive with a greater scope of activities than other telomere resolvases, the sequences mediating most of its unique properties have not yet been identified. The B. burgdorferi genome contains a total of 19 distinct hairpin sequences, all of which must be resolved by ResT (31.Tourand Y. Deneke J. Moriarty T.J. Chaconas G. J. Biol. Chem. 2009; 284: 7264-7272Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). These sequences can be classified into three groups based on the presence and positioning of the box 1 motif, which is a critical determinant of activity in phage and Borrelia telomere resolvases (see Fig. 1A) (21.Huang W.M. Joss L. Hsieh T. Casjens S. J. Mol. Biol. 2004; 337: 77-92Crossref PubMed Scopus (30) Google Scholar, 24.Deneke J. Ziegelin G. Lurz R. Lanka E. J. Biol. Chem. 2002; 277: 10410-10419Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 30.Tourand Y. Kobryn K. Chaconas G. Mol. Microbiol. 2003; 48: 901-911Crossref PubMed Scopus (35) Google Scholar). A box 1-like motif is also found in many of the hairpin telomeres sequenced to date (6.Casjens S.R. Gilcrease E.B. Huang W.M. Bunny K.L. Pedulla M.L. Ford M.E. Houtz J.M. Hatfull G.F. Hendrix R.W. J. Bacteriol. 2004; 186: 1818-1832Crossref PubMed Scopus (82) Google Scholar, 14.González A. Talavera A. Almendral J.M. Viñuela E. Nucleic Acids Res. 1986; 14: 6835-6844Crossref PubMed Scopus (101) Google Scholar, 32.Hinnebusch J. Barbour A.G. J. Bacteriol. 1991; 173: 7233-7239Crossref PubMed Scopus (109) Google Scholar, 33.Casjens S. Curr. Opin. Microbiol. 1999; 2: 529-534Crossref PubMed Scopus (70) Google Scholar, 34.Rybchin V.N. Svarchevsky A.N. Mol. Microbiol. 1999; 33: 895-903Crossref PubMed Scopus (73) Google Scholar, 35.Huang W.M. Robertson M. Aron J. Casjens S. J. Bacteriol. 2004; 186: 4134-4141Crossref PubMed Scopus (33) Google Scholar), although its function in telomere resolution is unknown. The box 1 consensus sequence (TAT(a/t)AT) closely resembles the −10/Pribnow box and TATA box consensus sequences of prokaryotic and eukaryotic promoters (TATAAT and TATA(a/t)A(a/t), respectively), which undergo transient deformations that predispose them to melting (36.Patel D.J. Kozlowski S.A. Weiss M. Bhatt R. Biochemistry. 1985; 24: 936-944Crossref PubMed Scopus (21) Google Scholar) and are intrinsically bent and anisotropically flexible (37.Davis N.A. Majee S.S. Kahn J.D. J. Mol. Biol. 1999; 291: 249-265Crossref PubMed Scopus (51) Google Scholar). Therefore, box 1 may facilitate nucleation of hairpin folding and/or may confer an intrinsic bend or flexibility to substrates that is important for the resolution reaction. B. burgdorferi ResT can resolve telomeres in which box 1 is located at positions 1 and 4 nucleotides away from the axis of symmetry (Type 1 and 2 telomeres, respectively), as well as AT-rich telomeres without a box 1 sequence (Type 3 telomeres) (see Fig. 1A) (30.Tourand Y. Kobryn K. Chaconas G. Mol. Microbiol. 2003; 48: 901-911Crossref PubMed Scopus (35) Google Scholar, 31.Tourand Y. Deneke J. Moriarty T.J. Chaconas G. J. Biol. Chem. 2009; 284: 7264-7272Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). B. burgdorferi ResT cleaves telomeres at a fixed position relative to the axis of symmetry, independent of the location of box 1 (30.Tourand Y. Kobryn K. Chaconas G. Mol. Microbiol. 2003; 48: 901-911Crossref PubMed Scopus (35) Google Scholar). Positioning of the enzyme for cleavage in all telomere types is most likely driven by sequence-specific interactions between ResT domains 2 (catalytic) and/or 3 (C-terminal) and a fixed element upstream of box 1 that is positioned 14 nucleotides from the axis of symmetry in all Borrelia telomeres (box 3 and adjacent nucleotides) (see Figs. 1A and 2) (26.Tourand Y. Lee L. Chaconas G. Mol. Microbiol. 2007; 64: 580-590Crossref PubMed Scopus (19) Google Scholar, 30.Tourand Y. Kobryn K. Chaconas G. Mol. Microbiol. 2003; 48: 901-911Crossref PubMed Scopus (35) Google Scholar, 31.Tourand Y. Deneke J. Moriarty T.J. Chaconas G. J. Biol. Chem. 2009; 284: 7264-7272Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In contrast, box 1 and axis-flanking nucleotides are not involved in high affinity and/or sequence-specific interactions with ResT and require the ResT N-terminal domain for full protection in DNase footprinting assays (26.Tourand Y. Lee L. Chaconas G. Mol. Microbiol. 2007; 64: 580-590Crossref PubMed Scopus (19) Google Scholar, 27.Bankhead T. Kobryn K. Chaconas G. Mol. Microbiol. 2006; 62: 895-905Crossref PubMed Scopus (24) Google Scholar). The most likely candidate for interactions with box 1 and axis-flanking nucleotides is a Borrelia-specific hairpin-binding region in the N terminus, which is thought to promote a pre-hairpinning step involving strand opening at the axis (38.Bankhead T. Chaconas G. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13768-13773Crossref PubMed Scopus (35) Google Scholar). ResT from the relapsing fever Borrelia species Borrelia hermsii exhibits a more restricted substrate usage pattern in vitro when compared with ResT from the Lyme disease pathogen B. burgdorferi (39.Tourand Y. Bankhead T. Wilson S.L. Putteet-Driver A.D. Barbour A.G. Byram R. Rosa P.A. Chaconas G. J. Bacteriol. 2006; 188: 7378-7386Crossref PubMed Scopus (28) Google Scholar). Specifically, B. hermsii ResT is unable to efficiently resolve a Type 2 telomere. Therefore, B. burgdorferi ResT appears to be a more permissive enzyme than its relapsing fever counterpart. In this study, we investigated the basis for permissive substrate usage by B. burgdorferi ResT. Using a library of chimeric B. hermsii/B. burgdorferi ResTs, we mapped the sequence determinants in B. burgdorferi ResT that confer the ability to resolve multiple Type 2 telomeres. Surprisingly, this approach indicated that Type 2 telomere resolution was crucially regulated by a single proline residue located in a small Borrelia-specific insertion in the central catalytic region of ResT. The proline at this position was conserved in the ResTs from all Lyme disease Borrelia species but in none of the ResTs from relapsing fever Borrelia species, which were unable to efficiently resolve Type 2 telomeres in vitro. This study has identified a specific residue in ResT responsible for permissive substrate usage patterns. All relapsing fever and avian Borrelia strains were generously provided by Tom Schwan. ResT coding sequences were cloned from B. anserina strain BA.2 (GCB802), B. parkeri strain RML (GCB803), B. recurrentis strain number 132, P6 (GCB804), and B. turicatae strain 91E135 (GCB801). GenBankTM accession numbers for ResT coding sequences are as follows: B. anserina (FJ882620), B. parkeri (FJ882621), B. recurrentis (FJ882622), and B. turicatae (FJ882623). ResT coding sequences were amplified from the appropriate genomic DNAs using primers containing NdeI and BamH1 sites (described in supplemental Table S2). PCR conditions for the 50-μl reactions were as follows: 1× Phusion High Fidelity (HF) buffer (New England Biolabs, Pickering, Ontario, Canada), 3% DMSO 3The abbreviation used is: DMSOdimethyl sulfoxide., 0.2 mm dNTPs, 0.02 units/μl Phusion DNA polymerase (New England Biolabs), 0.5 pmol/μl F primer, 0.5 pmol/μl R primer, 3 ng/μl genomic DNA template. PCR cycling conditions were as follows: 98 °C 45 s followed by 30 cycles of 98 °C, 10 s; 57 °C, 30 s; and 72 °C, 45 s and then 72 °C for 10 min. Appropriately sized PCR products were gel-purified using a Qiagen gel purification kit (Qiagen, Mississauga, Ontario, Canada) and then cloned using the ZeroBlunt®TOPO®PCR cloning kit (Invitrogen, Burlington, Ontario, Canada), all according to the manufacturer's instructions. Inserts were cut out of TOPO clones using NdeI/BamH1 (New England Biolabs) and cloned into NdeI/BamHI-digested pET15b. dimethyl sulfoxide. Constructs encoding chimeric proteins were built using site-directed mutagenesis or overlap extension PCR. Site-directed mutagenesis was used to introduce amino acid substitutions or to introduce unique restriction sites that were subsequently used for swapping sequences between B. burgdorferi and relapsing fever ResTs. The methods, templates, and primers used to build each construct are described in supplemental Table S2, and primer sequences are provided in supplemental Table S3. All constructs were sequenced before expression in E. coli. PCR conditions for the 50-μl reactions were as follows: 1× Phusion High Fidelity (HF) buffer (New England Biolabs), 3% DMSO, 0.1 mm dNTPs, 0.02 units/μl Phusion DNA polymerase (New England Biolabs), 0.4 pmol/μl F primer, 0.4 pmol/μl R primer, 1.5 ng/μl plasmid DNA template. The first three PCR cycles were performed separately for each primer (e.g. one tube for F primer, one tube for R primer). After the third cycle the contents of the F and R primer tubes were mixed, and PCR cycling was continued to completion. PCR cycling conditions were as follows: 98 °C 45 s followed by 25–30 cycles of 98 °C, 20 s; 65–68 °C, 15 s; 70–72 °C, 3 min and 30 s and then 72 °C for 7 min. PCR products were purified using the QIAQuick PCR purification kit, according to the manufacturer's instructions (Qiagen), and then template DNA was digested with DpnI (New England Biolabs). Purified, digested DNA was used directly to transform chemically competent DH5α. A description of the three-step PCR reaction and primer design is provided in supplemental Fig. S3. In the first step (25 PCR cycles), primers A and B were used to amplify the first approximately one-half of the chimera, and primers C and D were used to amplify the second half (e.g. B. burgdorferi ResT), in separate 50-μl reactions. Primers A and D contained 5′-NdeI and -BamHI sites, respectively. Primers B and C were partially complementary to one another and contained sequences that annealed to both B. burgdorferi and B. hermsii ResT coding sequences (60 °C of sequence complementarity for each). In the second PCR step (six PCR cycles), 2.5 μl of each of the two PCR products from Step 1 were mixed together directly (5-μl final reaction volume) and extended by DNA polymerase already present in the PCR mixture to generate a small amount of chimeric product containing all 1,347 nucleotides of the ResT coding sequence. In Step 3 (25 PCR cycles), 2.5 μl of full-length chimeric Step 2 product were amplified in a 50-μl PCR reaction, using primers A and D. PCR reaction conditions were as follows: 1× Phusion High Fidelity (HF) buffer (New England Biolabs), 3% DMSO, 0.1 mm dNTPs, 0.02 units/μl Phusion DNA polymerase (New England Biolabs), 0.1 pmol/μl F primer, 0.1 pmol/μl R primer, 1.5 ng/μl plasmid DNA template. PCR cycling conditions were as follows: 98 °C 3 min followed by 25 cycles of 98 °C, 15 s; 63 °C, 15 s; 72 °C, 30 s and then 72 °C for 7 min. Following PCR, products were gel-purified from agarose gels using the QIAQuick gel extraction kit (Qiagen). Cleaned products were cloned into pJET1/blunt using the GeneJET PCR cloning kit, according to the manufacturer's instructions (MBI Fermentas, Burlington, Ontario, Canada). NdeI/BamHI-flanked ResT coding sequences were cloned into NdeI/BamHI-digested pET15b. ResT expression and purification were performed as described previously (38.Bankhead T. Chaconas G. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13768-13773Crossref PubMed Scopus (35) Google Scholar) with the following exceptions. Bacteria were collected by centrifugation at 6,000 × g for 15 min at 4 °C. Pellets were resuspended in an EDTA-free buffer (25 mm Hepes-NaOH, pH 7.6). Resuspended cells were subjected to three freeze-thaw cycles following lysozyme treatment followed by ultracentrifugation for 45 min at 100,000 × g 4 °C. Columns for His tag affinity purification were prepared using 1 ml of nickel-nitrilotriacetic acid slurry (Qiagen). All purification buffers contained 0.5 m NaCl. Fractions were diluted to 50 ng/μl in elution buffer, and dithiothreitol was added to a final concentration of 1 mm. Previously described telomere substrates (30.Tourand Y. Kobryn K. Chaconas G. Mol. Microbiol. 2003; 48: 901-911Crossref PubMed Scopus (35) Google Scholar, 31.Tourand Y. Deneke J. Moriarty T.J. Chaconas G. J. Biol. Chem. 2009; 284: 7264-7272Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) were prepared from Qiagen midipreps (Qiagen), linearized with PstI (New England Biolabs), and cleaned by phenol extraction/ethanol precipitation. Twenty-μl telomere resolution reactions contained 25 mm Tris-HCl (pH 8.5), 100 mm NaCl, 1 mm EDTA, 100 μg/ml bovine serum albumin, 5 mm spermidine, 10 ng/μl PstI-linearized substrate DNA, and 10 ng/μl ResT. Time course reactions (140 μl) were incubated at 30 °C with 20-μl aliquots removed at 0, 1, 2, 4, 8, and 16 min. Reactions were performed in duplicate or triplicate and stopped by the addition of SDS to a final concentration of 0.2%. Samples were resolved on 20-cm 1% agarose gels in 1× Tris-acetate-EDTA (TAE) buffer at 100 V for 2 h. The gels were stained with ethidium bromide, and fluorescence of DNA bands was quantified using the AlphaInnotech software. The percentage of telomere resolution was determined by dividing the fluorescence of the reaction products by the total fluorescence (products plus substrate). Initial velocity values were calculated from the graphical plots of reaction kinetics. Statistical analyses of data were performed using Microsoft Excel and a two-tailed Student's t test with unequal variance. The methods used to identify and align telomere resolvase sequences are described in the legends for Fig. 2 and supplemental Fig. S2. The method used to thread ResT sequences onto the TelK structure is described in the legend for Fig. 4. Our previous work showed that B. burgdorferi (Lyme disease) and B. hermsii (relapsing fever) ResTs exhibit differences in substrate usage in vitro (39.Tourand Y. Bankhead T. Wilson S.L. Putteet-Driver A.D. Barbour A.G. Byram R. Rosa P.A. Chaconas G. J. Bacteriol. 2006; 188: 7378-7386Crossref PubMed Scopus (28) Google Scholar). Specifically, B. hermsii ResT resolves a Type 2 telomere very inefficiently when compared with B. burgdorferi, whereas both enzymes can resolve a Type 1 telomere efficiently (Fig. 1, A and B.) The basis for this species-specific difference in substrate usage is unknown. To determine whether ResTs from other relapsing fever Borrelia exhibit similar Type 2 telomere resolution defects, we cloned the ResT coding sequences from three other relapsing fever strains, B. parkeri, B. recurrentis, and B. turicatae, as well as the avian Borrelia species B. anserina. These sequences are shown in Fig. 2, together with the ResT sequences from other Lyme disease (Borrelia afzelii, Borrelia spielmanii, Borrelia garinii, and B. burgdorferi) and relapsing fever (B. hermsii and Borrelia duttonii) Borrelia species (39.Tourand Y. Bankhead T. Wilson S.L. Putteet-Driver A.D. Barbour A.G. Byram R. Rosa P.A. Chaconas G. J. Bacteriol. 2006; 188: 7378-7386Crossref PubMed Scopus (28) Google Scholar, 40.Fraser C.M. Casjens S. Huang W.M. Sutton G.G. Clayton R. Lathigra R. White O. Ketchum K.A. Dodson R. Hickey E.K. Gwinn M. Dougherty B. Tomb J.F. Fleischmann R.D. Richardson D. Peterson J. Kerlavage A.R. Quackenbush J. Salzberg S. Hanson M. van Vugt R. Palmer N. Adams M.D. Gocayne J. Weidman J. Utterback T. Watthey L. McDonald L. Artiach P. Bowman C. Garland S. Fuji C. Cotton M.D. Horst K. Roberts K. Hatch B. Smith H.O. Venter J.C. Nature. 1997; 39

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