Non-equivalent Interactions between Amino-terminal Domains of Neighboring λ Integrase Protomers Direct Holliday Junction Resolution

Lee, Sang Yeol; Radman‐Livaja, Marta; Warren, David J.; Aihara, Hideki; Ellenberger, Tom; Landy, Arthur

doi:10.1016/j.jmb.2004.10.068

Cited by 12 publications

(12 citation statements)

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“…If ResT could cleave hp telomeres outside the relatively safe context of a synapse, frequent double-strand break formation, loss of genetic material, and reduction in virulence or bacterial cell viability would result. It will be especially interesting, in future studies, to determine whether the regulation of ResT activity afforded by the requirements for in-line communication operates by allosteric domain swapping (used by the bacterial tyrosine recombinases that cleave in cis) or by contribution of the active site tyrosine in trans (as for Saccharomyces cerevisiae Flp) (41)(42)(43)(44)(45)(46).…”

Section: Discussionmentioning

confidence: 99%

Uncoupling the Chemical Steps of Telomere Resolution by ResT

Kobryn

Burgin²,

Chaconas

2005

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

Uncoupling the Chemical Steps of Telomere Resolution by ResT

Kobryn

Burgin²,

Chaconas

2005

Journal of Biological Chemistry

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“…The lambda coupler is a helix containing residues 64 to 74. Lee et al showed that it participates in HJ resolution by a protein-protein interaction (11). They found that substitution of either the D71 residue in the coupler or the R30 residue in the arm domain resulted in loss of HJ resolution activity.…”

Section: Resolution Of Holliday Junctions By Wild-type Intdot Sincementioning

confidence: 99%

“…If the strands that were exchanged initially to form the HJ are exchanged a second time, the HJ is resolved to re-form the substrates and the recombination reaction moves backward. If the strands that remained intact during HJ formation are exchanged, the HJ is resolved into recombinants and the recombination reaction moves forward (1,2,11,14,17).…”

mentioning

confidence: 99%

Resolution of Holliday Junction Recombination Intermediates by Wild-Type and Mutant IntDOT Proteins

Kim

Gardner

2011

J Bacteriol

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CTnDOT encodes an integrase that is a member of the tyrosine recombinase family. The recombination reaction proceeds by sequential sets of genetic exchanges between the attDOT site in CTnDOT and an attB site in the chromosome. The exchanges are separated by 7 base pairs in each site. Unlike most tyrosine recombinases, IntDOT exchanges sites that contain different DNA sequences between the exchange sites to generate Holliday junctions (HJs) that contain mismatched bases. We demonstrate that IntDOT resolves synthetic HJs in vitro. Holliday junctions that contain identical sequences between the exchange sites are resolved into both substrates and products, while HJs that contain mismatches are resolved only to substrates. This result implies that resolution of HJs to products requires the formation of a higher-order nucleoprotein complex with natural sites containing IntDOT. We also found that proteins with substitutions of residues (V95, K94, and K96) in a putative alpha helix at the junction of the N and CB domains (coupler region) were defective in resolving HJs. Mutational analysis of charged residues in the coupler and the N terminus of the protein did not provide evidence for a charge interaction between the regions of the protein. V95 may participate in a hydrophobic interaction with another region of IntDOT.IntDOT is an integrase encoded by CTnDOT, a Bacteroides integrative and conjugative element (ICE). IntDOT is a member of the tyrosine family of recombinases, which includes lambda integrase, FLP, CRE, and the XerC and XerD recombinases. Lambda Int and IntDOT are called factor-assisted tyrosine recombinases, because they use accessory factors to regulate directionality of their recombination reactions. These recombinases contain three DNA binding domains: the Nterminal arm-binding domain (N), the core binding domain (CB), and the catalytic domain (CAT). These recombinases bind to two different types of DNA sites: the arm-type sites and core-type sites. For lambda Int, it has been shown that the N domain binds to the arm-type sites and functions in the directionality and regulation of catalysis. The CB and CAT domains of lambda Int bind to the core-type sites and perform catalysis (1,15,17).Site-specific recombination reactions catalyzed by most tyrosine recombinases occur by two sequential sets of strand exchanges. The sites of exchange border a region of DNA, called the overlap region, that is usually the same in both recombination sites. The nucleoprotein complexes that perform the recombination reactions contain four recombinase monomers bound to the four core-type sites of the partner DNA substrates. Two of the recombinase monomers are catalytically active, while the other two monomers are inactive. The first strand exchanges are carried out by the two active recombinase monomers to form a four-way branch structure Holliday junction (HJ) intermediate. In order to execute the second set of strand exchanges to form products, the HJ intermediate undergoes a conformational change that results in the activ...

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“…The simplifying assumptions required for the interpretation of genetic data based on synthetic lethality (14) are now determined to be incompatible with the recent insights regarding flipped-out N-terminal domains (this work) and the nonequivalent interactions between N-terminal domains and linker regions of adjacent Int protomers (38).…”

Section: Discussionmentioning

confidence: 92%

“…This 2-fold symmetry explains why arm DNA stimulates resolution of only one partner pair within the Int HJ tetramer. Furthermore, this bias is influenced by nonequivalent interactions between the N-terminal domain of one Int and the linker region of an adjacent protomer within the tetrameric Int-HJ complex (38).…”

Section: Discussionmentioning

confidence: 99%