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

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 (1–6) and in bacteria themselves (7–10), including the Borrelia species that cause Lyme disease and relapsing fever (11, 12). A common solution to the end replication and protection problem in non-eukaryotes is the covalent sealing of DNA ends in the form of hairpins (2, 4–6, 10, 11, 13–16). 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, 18).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, 19–21). A growing number of candidate telomere resolvases have been identified in the genomes of eukaryotic viruses, phages, and bacteria (22, 23). Telomere resolvases are DNA cleavage and rejoining enzymes related to tyrosine recombinases and type 1B topoisomerases (19, 21, 22, 24, 25). 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, 19, 21, 24). 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); all but one of these residues are conserved in all telomere resolvases (22), implying that the basic catalytic mechanism underlying telomere resolution is conserved. However, telomere resolvase sequences vary substantially outside of the central catalytic region (25, 26), 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, 20, 21), it cannot efficiently resolve negatively supercoiled DNA (19, 27), 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), a function that is not shared with the phage telomere resolvase TelK (25). It can also synapse replicated telomeres and catalyze the formation of Holliday junctions (29). 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, 28). Finally, B. burgdorferi ResT can tolerate a surprising amount of variation in its substrate (30, 31), a feature that is not shared by phage telomere resolvases (21). 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). 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, 24, 30). A box 1-like motif is also found in many of the hairpin telomeres sequenced to date (6, 14, 32–35), 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) and are intrinsically bent and anisotropically flexible (37). 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.Open in a separate windowFIGURE 1.Species-specific resolution of Type I and 2 telomeres. A, a schematic showing the three types of hairpin telomere found on the linear replicons of the B. burgdorferi genome (see Ref. 31). The box 1 sequence in Type 1 and 2 telomeres is situated 1 and 4 nucleotides away from the axis of symmetry, respectively, whereas Type 3 telomeres contain no clear box 1. B, a schematic illustrating the telomere resolution reaction substrate and products is shown along with two ethidium bromide-stained agarose gels showing telomere resolution assays. The gels show resolution kinetics for B. burgdorferi and B. hermsii ResT on Type 1 and 2 telomeres (plasmid substrates pYT1/lp17L and pYT92/chromL, respectively).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, 31). B. burgdorferi ResT cleaves telomeres at a fixed position relative to the axis of symmetry, independent of the location of box 1 (30). 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 ​and2)2) (26, 30, 31). 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, 27). 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).Open in a separate windowFIGURE 2.Alignment of 11 Borrelia ResT sequences. Shown is ClustalW2 alignment of ResT amino acid sequences from five Lyme disease Borrelia species (B. afzelii, B. spielmanii, B. valaisiana, B. garinii, and B. burgdorferi), five relapsing fever Borrelia species (B. turicatae, B. parkeri, B. hermsii, B. recurrentis, and B. duttonii), and one avian Borrelia species (B. anserina) (generated using ClustalW2 from the EBI web site) (19, 39–42, 48, 49). The sequences for B. anserina, B. parkeri, and B. turicatae ResTs are reported for the first time in this study (respective GenBank accession numbers are {"type":"entrez-nucleotide","attrs":{"text":"FJ882620","term_id":"242263541","term_text":"FJ882620"}}FJ882620, {"type":"entrez-nucleotide","attrs":{"text":"FJ882621","term_id":"242263543","term_text":"FJ882621"}}FJ882621, and {"type":"entrez-nucleotide","attrs":{"text":"FJ882623","term_id":"242263547","term_text":"FJ882623"}}FJ882623). Sequences are arranged in order of similarity to neighboring sequences and are colored in JalView using the Zappo coloring scheme for identifying amino acids with similar physicochemical properties (50). Only residues that are identical in 100% of ResTs are indicated by colored shading. Arrows above the alignment indicate ResT domain boundaries identified by chymotrypsin digest, sequence comparison with other proteins, and HHsenser predictions (26, 51). The hairpin-binding motif found in cut-and-paste transposases is indicated beneath the alignment by white text on a black background (38). The positions corresponding to the active site residues in tyrosine recombinases, type IB topoisomerases, and TelK are indicated by blue asterisks below the sequence, with the active site tyrosine nucleophile at position 335 marked by a red asterisk (22, 25). The ringed black dot below position 326 indicates an amino acid in the active site region that differs in Lyme disease and relapsing fever ResTs. Sequences above the black line drawn between B. burgdorferi and B. turicatae are from Lyme disease Borrelia species; sequences below the black line are from relapsing fever Borrelia species. The ResT sequence from the avian Borrelia species B. anserina is shown at bottom.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). 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.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.Infection with Helicobacter pylori strains bearing cagA (cytotoxin-associated gene A)-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (1–3). The cagA gene is located within a 40-kb DNA fragment, termed the cag pathogenicity island, which is specifically present in the genome of cagA-positive H. pylori strains (4–6). In addition to cagA, there are ∼30 genes in the cag pathogenicity island, many of which encode a bacterial type IV secretion system that delivers the cagA-encoded CagA protein into gastric epithelial cells (7–10). Upon delivery into gastric epithelial cells, CagA is localized to the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala motifs by Src family kinases or c-Abl kinase (11–14). The C-terminal Glu-Pro-Ile-Tyr-Ala-containing region of CagA is noted for the structural diversity among distinct H. pylori isolates. Oncogenic potential of CagA has recently been confirmed by a study showing that systemic expression of CagA in mice induces gastrointestinal and hematological malignancies (15).When expressed in gastric epithelial cells, CagA induces morphological transformation termed the hummingbird phenotype, which is characterized by the development of one or two long and thin protrusions resembling the beak of the hummingbird. It has been thought that the hummingbird phenotype is related to the oncogenic action of CagA (7, 16–19). Pathophysiological relevance for the hummingbird phenotype in gastric carcinogenesis has recently been provided by the observation that infection with H. pylori carrying CagA with greater ability to induce the hummingbird phenotype is more closely associated with gastric carcinoma (20–23). Elevated motility of hummingbird cells (cells showing the hummingbird phenotype) may also contribute to invasion and metastasis of gastric carcinoma.In host cells, CagA interacts with the SHP-2 phosphatase, C-terminal Src kinase, and Crk adaptor in a tyrosine phosphorylation-dependent manner (16, 24, 25) and also associates with Grb2 adaptor and c-Met in a phosphorylation-independent manner (26, 27). Among these CagA targets, much attention has been focused on SHP-2 because the phosphatase has been recognized as a bona fide oncoprotein, gain-of-function mutations of which are found in various human malignancies (17, 18, 28). Stable interaction of CagA with SHP-2 requires CagA dimerization, which is mediated by a 16-amino acid CagA-multimerization (CM)² sequence present in the C-terminal region of CagA (29). Upon complex formation, CagA aberrantly activates SHP-2 and thereby elicits sustained ERK MAP kinase activation that promotes mitogenesis (30). Also, CagA-activated SHP-2 dephosphorylates and inhibits focal adhesion kinase (FAK), causing impaired focal adhesions. It has been shown previously that both aberrant ERK activation and FAK inhibition by CagA-deregulated SHP-2 are involved in induction of the hummingbird phenotype (31).Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) is an evolutionally conserved serine/threonine kinase originally isolated in C. elegans (32–34). Mammalian cells possess four structurally related PAR1 isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4 (35–37). Among these, PAR1a, PAR1b, and PAR1c are expressed in a variety of cells, whereas PAR1d is predominantly expressed in neural cells (35, 37). These PAR1 isoforms phosphorylate microtubule-associated proteins (MAPs) and thereby destabilize microtubules (35, 38), allowing asymmetric distribution of molecules that are involved in the establishment and maintenance of cell polarity.In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basolateral polarity (39, 40). This CagA activity involves the interaction of CagA with PAR1b/MARK2 (19, 41). CagA directly binds to the kinase domain of PAR1b in a tyrosine phosphorylation-independent manner and inhibits the kinase activity. Notably, CagA binds to PAR1b via the CM sequence (19). Because PAR1b is present as a dimer in cells (42), CagA may passively homodimerize upon complex formation with the PAR1 dimer via the CM sequence, and this PAR1-directed CagA dimer would form a stable complex with SHP-2 through its two SH2 domains.Because of the critical role of CagA in gastric carcinogenesis (7, 16–19), it is important to elucidate the molecular basis underlying the morphogenetic activity of CagA. In this study, we investigated the role of PAR1 isoforms in induction of the hummingbird phenotype by CagA, and we obtained evidence that CagA-mediated inhibition of PAR1 kinases contributes to the development of the morphological change by perturbing microtubules and non-muscle myosin II.     

Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]