[Show abstract][Hide abstract] ABSTRACT: Author Summary
Linear chromosomes capped by hairpin telomeres are widespread in prokaryotes and are found in important bacterial pathogens. However, three-dimensional structure of the hairpin telomere, as well as the molecular mechanisms underlying its generation, has remained poorly understood. In this work, we investigated how the enzyme responsible for generating the bacterial hairpin telomeres (protelomerase, also known as telomere resolvase) transforms a linear double-stranded DNA molecule into sharp hairpin turns. Our X-ray crystallographic and biochemical data collectively suggest that protelomerase employs a multistep DNA strand-refolding mechanism as described below. Protelomerase first cleaves both strands of a double-helical DNA substrate and reshapes the DNA strands into a transition state conformation (refolding intermediate) stabilized by specific protein–DNA and DNA–DNA interactions including noncanonical (non-Watson–Crick) base-pairs. The DNA strands are then refolded into extremely compact hairpin products, stabilized by a set of interactions distinct from those stabilizing the refolding intermediate. We believe that an enzyme “catalyzing” not only the chemical reactions of DNA strand cutting/rejoining but also the ordered transition between different DNA conformations to guide refolding of the DNA strand is a novel concept, and we suspect that similar mechanisms may be employed by other enzymes involved in conformational changes/refolding of biological macromolecules.
[Show abstract][Hide abstract] ABSTRACT: Sequence-specific DNA-binding proteins must quickly and reliably localize specific target sites on DNA. This search process has been well characterized for monomeric proteins, but it remains poorly understood for systems that require assembly into dimers or oligomers at the target site. We present a single-molecule study of the target-search mechanism of protelomerase TelK, a recombinase-like protein that is only active as a dimer. We show that TelK undergoes 1D diffusion on non-target DNA as a monomer, and it immobilizes upon dimerization even in the absence of a DNA target site. We further show that dimeric TelK condenses non-target DNA, forming a tightly bound nucleoprotein complex. Together with theoretical calculations and molecular dynamics simulations, we present a novel target-search model for TelK, which may be generalizable to other dimer and oligomer-active proteins.
Nucleic Acids Research 12/2012; 41(4). DOI:10.1093/nar/gks1345 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.
[Show abstract][Hide abstract] ABSTRACT: Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi ∼900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short ≤20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.
PLoS ONE 03/2012; 7(3):e33280. DOI:10.1371/journal.pone.0033280 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein-DNA interactions are essential to cellular processes, many of
which require proteins to recognize a specific DNA target-site. This
search process is well-documented for monomeric proteins, but not as
well understood for systems that require dimerization at the target site
for activity. We present a single-molecule study of the target-search
mechanism of Protelomerase TelK, a recombinase-like protein that is only
active as a dimer. We observe that TelK undergoes 1D diffusion on
non-target DNA as a monomer, as expected, but becomes immobile on DNA as
a dimer or oligomer despite the absence of its target site. We further
show that TelK condenses non-target DNA upon dimerization, forming a
tightly bound nucleo-protein complex. Together with simulations, our
results suggest a search model whereby monomers diffuse along DNA, and
subsequently dimerize to form an active complex on target DNA. These
results show that target-finding occurs faster than nonspecific
dimerization at biologically relevant protein concentrations. This model
may provide insights into the search mechanisms of proteins that are
active as multimeric complexes for a more accurate and comprehensive
model for the target-search process by sequence specific proteins.
[Show abstract][Hide abstract] ABSTRACT: We report the genome sequence of Bacillus subtilis phage SPO1. The unique genome sequence is 132,562 bp long, and DNA packaged in the virion (the chromosome) has a 13,185-bp terminal redundancy, giving a total of 145,747 bp. We predict 204 protein-coding genes and 5 tRNA genes, and we correlate these findings with the extensive body of investigations of SPO1, including studies of the functions of the 61 previously defined genes and studies of the virion structure. Sixty-nine percent of the encoded proteins show no similarity to any previously known protein. We identify 107 probable transcription promoters; most are members of the promoter classes identified in earlier studies, but we also see a new class that has the same sequence as the host sigma K promoters. We find three genes encoding potential new transcription factors, one of which is a distant homologue of the host sigma factor K. We also identify 75 probable transcription terminator structures. Promoters and terminators are generally located between genes and together with earlier data give what appears to be a rather complete picture of how phage transcription is regulated. There are complete genome sequences available for five additional phages of Gram-positive hosts that are similar to SPO1 in genome size and in composition and organization of genes. Comparative analysis of SPO1 in the context of these other phages yields insights about SPO1 and the other phages that would not be apparent from the analysis of any one phage alone. These include assigning identities as well as probable functions for several specific genes and inferring evolutionary events in the phages' histories. The comparative analysis also allows us to put SPO1 into a phylogenetic context. We see a pattern similar to what has been noted in phage T4 and its relatives, in which there is minimal successful horizontal exchange of genes among a "core" set of genes that includes most of the virion structural genes and some genes of DNA metabolism, but there is extensive horizontal transfer of genes over the remainder of the genome. There is a correlation between genes in rapid evolutionary flux through these genomes and genes that are small.
[Show abstract][Hide abstract] ABSTRACT: Protein-DNA interactions lie at the heart of many essential cellular processes such as replication, recombination, and repair. Recent advances in optical ``tweezers'' have made it possible to resolve motions on the scale of a single base pair of DNA, 3.4å. High-resolution optical traps have the potential to reveal these interactions at their fundamental length scales and should reveal how certain proteins bind to DNA or recognize target sequences. Telomerases are enzymes that have been actively studied in various organisms because of their fundamental involvement with both cancer and aging^1. Protelomerase TelK is an enzyme responsible for forming closed DNA hairpin ends in linear DNA. TelK is not an ATP dependant enzyme, which is surprising given the degree of DNA distortion accomplished by the enzyme, and the large energy barrier intrinsic in DNA hairpin formation. Therefore, our focus is on TelK mutants lacking their c-terminal domain, and TelK YF mutants lacking their tyrosine active site amino acid. Preliminary data have shown remarkable differences in protein binding and unbinding forces caused by the removal of a single oxygen atom from a 73 kDa protein. Further measurements using high-resolution optical tweezers should provide fundamental insights into the nature and importance of the electrostatic interactions between TelK and its DNA substrate. 1. Shay, J. et al. Rad. Res. 155, 188 (2001)  Huang, W. et al. Mol. Cell. 27, 901 (2007).
[Show abstract][Hide abstract] ABSTRACT: Half the ribosomes translating the mRNA for phage T4 gene 60 topoisomerase subunit bypass a 50 nucleotide coding gap between codons 46 and 47. The pairing of codon 46 with its cognate peptidyl-tRNA anticodon dissociates, and following mRNA slippage, peptidyl-tRNA re-pairs to mRNA at a matched triplet 5' adjacent to codon 47, where translation resumes. Here, in studies with gene 60 cassettes, it is shown that the peptidyl-tRNA anticodon does not scan the intervening sequence for potential complementarity. However, certain coding gap mutants allow peptidyl-tRNA to scan sequences in the bypassed segment. A model is proposed in which the coding gap mRNA enters the ribosomal A-site and forms a structure that precludes peptidyl-tRNA scanning of its sequence. Dissipation of this RNA structure, together with the contribution of 16S rRNA anti-Shine-Dalgarno sequence pairing with GAG, facilitates peptidyl-tRNA re-pairing to mRNA.
The EMBO Journal 10/2008; 27(19):2533-44. DOI:10.1038/emboj.2008.170 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The termini of linear chromosomes are protected by specialized DNA structures known as telomeres that also facilitate the complete replication of DNA ends. The simplest type of telomere is a covalently closed DNA hairpin structure found in linear chromosomes of prokaryotes and viruses. Bidirectional replication of a chromosome with hairpin telomeres produces a catenated circular dimer that is subsequently resolved into unit-length chromosomes by a dedicated DNA cleavage-rejoining enzyme known as a hairpin telomere resolvase (protelomerase). Here we report a crystal structure of the protelomerase TelK from Klebsiella oxytoca phage varphiKO2, in complex with the palindromic target DNA. The structure shows the TelK dimer destabilizes base pairing interactions to promote the refolding of cleaved DNA ends into two hairpin ends. We propose that the hairpinning reaction is made effectively irreversible by a unique protein-induced distortion of the DNA substrate that prevents religation of the cleaved DNA substrate.
[Show abstract][Hide abstract] ABSTRACT: Bacteria possess two closely related yet functionally distinct essential type IIA topoisomerases (Topos). DNA gyrase supports replication and transcription with its unique supercoiling activity, whereas Topo IV preferentially relaxes (+) supercoils and is a decatenating enzyme required for chromosome segregation. Here we report the crystal structure of the C-terminal domain of Topo IV ParC subunit (ParC-CTD) from Bacillus stearothermophilus and provide a structure-based explanation for how Topo IV and DNA gyrase execute distinct activities. Although the topological connectivity of ParC-CTD is similar to the recently determined CTD structure of DNA gyrase GyrA subunit (GyrA-CTD), ParC-CTD surprisingly folds as a previously unseen broken form of a six-bladed beta-propeller. Propeller breakage is due to the absence of a DNA gyrase-specific GyrA box motif, resulting in the reduction of curvature of the proposed DNA binding region, which explains why ParC-CTD is less efficient than GyrA-CTD in mediating DNA bending, a difference that leads to divergent activities of the two homologous enzymes. Moreover, we found that the topology of the propeller blades observed in ParC-CTD and GyrA-CTD can be achieved from a concerted beta-hairpin invasion-induced fold change event of a canonical six-bladed beta-propeller; hence, we proposed to name this new fold as "hairpin-invaded beta-propeller" to highlight the high degree of similarity and a potential evolutionary linkage between them. The possible role of ParC-CTD as a geometry facilitator during various catalytic events and the evolutionary relationships between prokaryotic type IIA Topos have also been discussed according to these new structural insights.
[Show abstract][Hide abstract] ABSTRACT: Spirochetes in the genus Borrelia carry a linear chromosome and numerous linear plasmids that have covalently closed hairpin telomeres. The overall organization
of the large chromosome of Borrelia burgdorferi appears to have been quite stable over recent evolutionary time; however, a large fraction of natural isolates carry differing
lengths of DNA that extend the right end of the chromosome between about 7 and 20 kbp relative to the shortest chromosomes.
We present evidence here that a rather recent nonhomologous recombination event in the B. burgdorferi strain Sh-2-82 lineage has replaced its right chromosomal telomere with a large portion of the linear plasmid lp21, which
is present in the strain B31 lineage. At least two successive rounds of addition of linear plasmid genetic material to the
chromosomal right end appear to have occurred at the Sh-2-82 right telomere, suggesting that this is an evolutionary mechanism
by which plasmid genetic material can become part of the chromosome. The unusual nonhomologous nature of this rearrangement
suggests that, barring horizontal transfer, it can be used as a unique genetic marker for this lineage of B. burgdorferi chromosomes.
Journal of Bacteriology 08/2004; 186(13):4134-41. DOI:10.1128/JB.186.13.4134-4141.2004 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protelomerases are enzymes responsible for the generation of closed hairpin ends in linear DNA. It is proposed that they use a breaking-and-rejoin type mechanism to affect DNA rearrangement on specific DNA sequences. In doing so, one strand turns around and becomes the complementary strand. Using the purified enzyme from the Escherichia coli phage N15 and the Klebsiella phage φKO2 and synthetic oligonucleotide substrates, we directly demonstrate the location where the cutting/re-ligation occurs. We identified a pair of transient staggered cleavages six base-pairs apart centered around the axis of dyad symmetry of the target site. Two molecules of the protelomerase form a pair of protein-linked DNA intermediates at each 3′ end of the cleaved openings leaving a 5′-OH. Then, in a process not yet clearly defined, the partners of the two initial openings are exchanged, and the transient breaks are resealed to generate hairpin ends. The formation of 3′-covalent DNA-protein intermediates is a hallmark of the topoisomerase IB type reaction, and we have thus shown experimentally that protelomerase is a member of the tyrosine-recombinase superfamily. In addition, by introducing single nicks in the substrates as perturbation, we found that the integrity of the nucleotide chain 4 bp away from the cutting site as well as this nucleotide's complementary location on the stem if the strands were to fold into a cruciform structure are required for activity, suggesting that these locations may be important substrate-protein contacts. We determined that N15 and φKO2 protelomerases are monomers in solution and two molecules are needed to interact with the substrate to form two closed hairpin products. The target sites of protelomerases invariably consist of inverted repeats. Comparative studies using the related target sites of different protelomerases suggest that these proteins may require both sequence-specific and structure (possibly cruciform)-specific recognition for activity.
[Show abstract][Hide abstract] ABSTRACT: Temperate bacteriophages with plasmid prophages are uncommon in nature, and of these only phages N15 and PY54 are known to
have a linear plasmid prophage with closed hairpin telomeres. We report here the complete nucleotide sequence of the 51,601-bp
Klebsiella oxytoca linear plasmid pKO2, and we demonstrate experimentally that it is also a prophage. We call this bacteriophage φKO2. An analysis
of the 64 predicted φKO2 genes indicate that it is a fairly close relative of phage N15; they share a mosaic relationship
that is typical of different members of double-stranded DNA tailed-phage groups. Although the head, tail shaft, and lysis
genes are not recognizably homologous between these phages, other genes such as the plasmid partitioning, replicase, prophage
repressor, and protelomerase genes (and their putative targets) are so similar that we predict that they must have nearly
identical DNA binding specificities. The φKO2 virion is unusual in that its phage λ-like tails have an exceptionally long
(3,433 amino acids) central tip tail fiber protein. The φKO2 genome also carries putative homologues of bacterial dinI and umuD genes, both of which are involved in the host SOS response. We show that these divergently transcribed genes are regulated
by LexA protein binding to a single target site that overlaps both promoters.
Journal of Bacteriology 04/2004; 186(6):1818-32. DOI:10.1128/JB.186.6.1818-1832.2004 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non-functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes ≥300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.
[Show abstract][Hide abstract] ABSTRACT: Bacteria of the spirochaete genus Borrelia have linear chromosomes about 950 kbp in size. We report here that these linear chromosomes have covalently closed hairpin structures at their termini that are similar but not identical to those reported for linear plasmids carried by these organisms. Nucleotide sequence analysis of the chromosomal telomeric regions indicates that unique, apparently functional genes lie within a few hundred bp of each of the telomeres, and that there is an imperfect 26 bp inverted repeat at the two telomeres. In addition, we characterize a major chromosomal length polymorphism within the right telomeric regions of various Borrelia isolates, and show that sequences similar to those near the right telomere are often found on linear plasmids in B. burgdorferi (sensu stricto) isolates from nature. Sequences similar to a number of other regions of the chromosome, including those near the left telomere, were not found on B. burgdorferi plasmids. These observations suggest that there has been historical exchange of genetic information between the linear plasmids and the right end of the linear chromosome.
[Show abstract][Hide abstract] ABSTRACT: A type-II toposiomerase (Topo-IV) encoded by the parC and parE genes in Escherichia coli and Salmonella typhimurium is thought to be involved in cell septation and in the decatenation of newly replicated chromosomes. We have identified parC and parE homologs in the pleomorphic, wall-less organism Mycoplasma genitalium. Since the mechanics of cell septation in conventional eubacterial species is believed to be mediated by cell-wall constituents, there is no clear understanding of what coordinates that process in wall-less species. The presence of par genes in this bacterium, which has the smallest genome of any free-living organism, suggests that Topo-IV has been evolutionarily conserved because of an essential role in mediating cell division.
[Show abstract][Hide abstract] ABSTRACT: Type II DNA topoisomerases are essential and ubiquitous DNA metabolic enzymes that alter DNA topology. Eubacteria have two indispensable type II DNA topoisomerases, DNA gyrase encoded by gyrB and gyrA and topoisomerase IV encoded by parE and parC. These genes belong to a single family whose members span both eukaryotes and prokaryotes. The highly conserved motifs in these genes provide a rationale for the design of universal primers used in the polymerase chain reaction in order to systematically generate a data set suitable for bacterial diversity studies at the macro-diversity level, as well as at the micro-diversity level displaying individual species and isolates. This family of genes is the subject of intensive biochemical and genetic analyses, which provide an opportunity for comprehensive understanding of sequence conservation and variability and their relationship to function. These genes are ideally suited for microbial identification and biodiversity analyses.
[Show abstract][Hide abstract] ABSTRACT: The nucleotide sequence of the Borrelia burgdorferi (Bb) Sh-2-82 recA gene has been determined using PCR-based approaches without the construction of a genomic library. The gene should encode a protein of 365 amino acids which is highly homologous to other known RecA proteins. It represents a new homolog from a distinct phylogenetic branch of eubacteria. Although, previous reports concluded that recA is absent from Bb, the identification presented here conclusively shows its presence and reaffirms the ubiquity of RecA in prokaryotes.