[show abstract][hide abstract] ABSTRACT: Hairpin telomeres of bacterial linear chromosomes are generated by a DNA cutting-rejoining enzyme protelomerase. Protelomerase resolves a concatenated dimer of chromosomes as the last step of chromosome replication, converting a palindromic DNA sequence at the junctions between chromosomes into covalently closed hairpins. The mechanism by which protelomerase transforms a duplex DNA substrate into the hairpin telomeres remains largely unknown. We report here a series of crystal structures of the protelomerase TelA bound to DNA that represent distinct stages along the reaction pathway. The structures suggest that TelA converts a linear duplex substrate into hairpin turns via a transient strand-refolding intermediate that involves DNA-base flipping and wobble base-pairs. The extremely compact di-nucleotide hairpin structure of the product is fully stabilized by TelA prior to strand ligation, which drives the reaction to completion. The enzyme-catalyzed, multistep strand refolding is a novel mechanism in DNA rearrangement reactions.
[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; · 8.28 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.
Journal of Biological Chemistry 05/2012; 287(30):25551-63. · 4.65 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: 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 01/2012; 7(3):e33280. · 3.73 Impact Factor
[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.
Journal of Molecular Biology 05/2009; 388(1):48-70. · 3.91 Impact Factor
[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. · 9.82 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: 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. · 3.19 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 phiKO2 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 phiKO2 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.
Journal of Molecular Biology 04/2004; 337(1):77-92. · 3.91 Impact Factor
[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 phiKO2. An analysis of the 64 predicted phiKO2 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 phiKO2 virion is unusual in that its phage lambda-like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The phiKO2 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. · 3.19 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.