The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products.

The bacterial transposon Tn7 translocates by a cut and paste mechanism: excision from the donor site results from double-strand breaks at each end of Tn7 and target insertion results from joining of the exposed 3'' Tn7 tips to the target DNA. Through site-directed mutagenesis of the Tn7-encoded transposition proteins TnsA and TnsB, we demonstrate that the Tn7 transposase is a heteromeric complex of these proteins, each protein executing different DNA processing reactions. TnsA mediates DNA cleavage reactions at the 5'' ends of Tn7, and TnsB mediates DNA breakage and joining reactions at the 3'' ends of Tn7. Thus the double-strand breaks that underlie Tn7 excision result from a collaboration between two active sites, one in TnsA and one in TnsB; the same (or a closely related) active site in TnsB also mediates the subsequent joining of the 3'' ends to the target. Both TnsA and TnsB appear to be members of the retroviral integrase superfamily: mutation of their putative DD(35)E motifs blocks catalytic activity. Recombinases of this class require a divalent metal cofactor that is thought to interact with these acidic residues. Through analysis of the metal ion specificity of a TnsA mutant containing a sulfur (cysteine) substitution, we provide evidence that a divalent metal actually interacts with these acidic amino acids.     

The three methyl-CpG-binding domains of AtMBD7 control its subnuclear localization and mobility

Three methyl-CpG-binding domain (MBD) proteins in Arabidopsis, AtMBD5, AtMBD6, and AtMBD7, are functional in binding methylated CpG dinucleotides in vitro and localize to the highly CpG-methylated chromocenters in vivo. These proteins differ, however, in their subnuclear localization pattern; AtMBD5 and AtMBD6, each containing a single MBD motif, show preference for two perinucleolar chromocenters, whereas AtMBD7, a naturally occurring poly-MBD protein containing three MBD motifs, localizes to all chromocenters. Here we studied the significance of multiple MBD motifs for subnuclear localization and mobility in living cells. We found that the number of MBD motifs determines the subnuclear localization of the MBD protein. Furthermore, live kinetic experiments showed that AtMBD7-green fluorescent protein (GFP) has lower mobility than AtMBD5-GFP and AtMBD6-GFP, which is conferred by cooperative activity of its three MBD motifs. Thus, the number of MBD motifs appears to affect not only binding affinity and mobility within the nucleus, but also the subnuclear localization of the protein. Our results suggest that poly-MBD proteins can directly affect chromatin structure by inducing intra- and inter-chromatin compaction via bridging over multiple methylated CpG sites.     

Unexpected structural diversity in DNA recombination: the restriction endonuclease connection

Transposition requires a coordinated series of DNA breakage and joining reactions. The Tn7 transposase contains two proteins: TnsA, which carries out DNA breakage at the 5' ends of the transposon, and TnsB, which carries out breakage and joining at the 3' ends of the transposon. TnsB is a member of the retroviral integrase superfamily whose hallmark is a conserved DDE motif. We report here the structure of TnsA at 2.4 A resolution. Surprisingly, the TnsA fold is that of a type II restriction endonuclease. Thus, Tn7 transposition involves a collaboration between polypeptides, one containing a DDE motif and one that does not. This result indicates that the range of biological processes that utilize restriction enzyme-like folds also includes DNA transposition.     

Formation of a nucleoprotein complex containing Tn7 and its target DNA regulates transposition initiation

Tn7 insertion into its specific target site, attTn7, is mediated by the proteins TnsA, TnsB, TnsC and TnsD. The double-strand breaks that separate Tn7 from the donor DNA require the Tns proteins, the transposon and an attTn7 target DNA, suggesting that a prerequisite for transposition is the formation of a nucleoprotein complex containing TnsABC+D, and these DNAs. Here, we identify a TnsABC+D transposon-attTn7 complex, and demonstrate that it is a transposition intermediate. We demonstrate that an interaction between TnsB, the transposase subunit that binds to the transposon ends, and TnsC, the target DNA-binding protein that controls the activity of the transposase, is essential for assembly of the TnsABC+D transposon-attTn7 complex. We also show that certain TnsB residues are required for recombination because they mediate a TnsB-TnsC interaction critical to formation of the TnsABC+D transposon-attTn7 complex. We demonstrate that TnsA, the other transposase subunit, which also interacts with TnsC, greatly stabilizes the TnsABC+D transposon-attTn7 complex. Thus multiple interactions between the transposase subunits, TnsA and TnsB, and the target-binding transposase activator, TnsC, control Tn7 transposition.     

The carboxy-terminal portion of TnsC activates the Tn7 transposase through a specific interaction with TnsA

Tn7 transposition requires the assembly of a nucleoprotein complex containing four self-encoded proteins, transposon ends, and target DNA. Within this complex, TnsC, the molecular switch that regulates transposition, and TnsA, one part of the transposase, interact directly. Here, we demonstrate that residues 504-555 of TnsC are responsible for TnsA/TnsC interaction. The crystal structure of the TnsA/TnsC(504-555) complex, resolved to 1.85 A, illustrates the burial of a large hydrophobic patch on the surface of TnsA. One consequence of sequestering this patch is a marked increase in the thermal stability of TnsA as shown by differential scanning calorimetry. A model based on the complex structure suggested that TnsA and a slightly longer version of the cocrystallized TnsC fragment (residues 495-555) might cooperate to bind DNA, a prediction confirmed using gel mobility shift assays. Donor DNA binding by the TnsA/TnsC(495-555) complex is correlated with the activation of the TnsAB transposase, as measured by double-stranded DNA cleavage assays, demonstrating the importance of the TnsA/TnsC interaction in affecting Tn7 transposition.     

Isolation and characterization of Tn7 transposase gain-of-function mutants: a model for transposase activation

Tn7 transposition has been hypothesized to require a heteromeric transposase formed by two Tn7-encoded proteins, TnsA and TnsB, and accessory proteins that activate the transposase when they are associated with an appropriate target DNA. This study investigates the mechanism of Tn7 transposase activation by isolation and analysis of transposase gain-of-function mutants that are active in the absence of these accessory proteins. This work shows directly that TnsA and TnsB are essential and sufficient components of the Tn7 transposase and also provides insight into the signals that activate the transposase. We also describe a protein-protein interaction between TnsA and TnsC, a regulatory accessory protein, that is likely to be critical for transposase activation.     

Dual DNA unwinding activities of the Rothmund–Thomson syndrome protein,RECQ4

Human RECQ helicases have been linked to distinct clinical diseases with increased cancer rates and premature ageing. All RECQ proteins, except RECQ4, have been shown to be functional helicases. Mutations in RECQ4 lead to Rothmund–Thomson syndrome (RTS), and mouse models reveal that the conserved helicase motifs are required for avoidance of RTS. Furthermore, the amino (N) terminus of RECQ4 shares homology with yeast DNA replication initiation factor, Sld2, and is vital for embryonic development. Here, in contrast to previous reports, we show that RECQ4 exhibits DNA helicase activity. Importantly, two distinct regions of the protein, the conserved helicase motifs and the Sld2‐like N‐terminal domain, each independently promote ATP‐dependent DNA unwinding. Taken together, our data provide the first biochemical clues underlying the molecular function of RECQ4 in DNA replication and genome maintenance.     

The relationship between periodic dinucleotides and the nucleosomal DNA deformation revealed by normal mode analysis

Nucleosomes, which contain DNA and proteins, are the basic unit of eukaryotic chromatins. Polymers such as DNA and proteins are dynamic, and their conformational changes can lead to functional changes. Periodic dinucleotide patterns exist in nucleosomal DNA chains and play an important role in the nucleosome structure. In this paper, we use normal mode analysis to detect significant structural deformations of nucleosomal DNA and investigate the relationship between periodic dinucleotides and DNA motions. We have found that periodic dinucleotides are usually located at the peaks or valleys of DNA and protein motions, revealing that they dominate the nucleosome dynamics. Also, a specific dinucleotide pattern CA/TG appears most frequently.     

Statistical evaluation of the coding capacity of complementary DNA strands

Two independent methods are used to evaluate the protein-coding information content in different classes of DNA sequences. The first method allows to evaluate the statistical relevance of finding unidentified reading frames, longer than 100 codons, on both DNA strands of: a) 117 DNA sequences that code for 142 nuclear proteins; b) 39 stable RNA coding sequences and c) 36 other DNA sequences which include regulatory and as yet unknown function sequences. The finding of 50 reading frames longer than 100 codons (complementary inverted proteins or c.i.p. genes) located on the DNA strand complementary to the protein-coding one is drastically in excess of the number predicted by chance alone. An independent method (testcode) applied to c.i.p. gene sequences, which assigns the probability of coding to a given sequence, predicts that more than 50% of these genes are translated in a functional product. These analyses indicate the existence of a new class of protein-coding genes, located on the DNA sequences complementary to the protein-coding DNA strand.     

Densely methylated DNA islands in mammalian chromosomal replication origins.

Densely methylated DNA sequence islands, designated DMIs, have been observed in two Chinese hamster cell chromosomal replication origins by using a PCR-based chemical method of detection. One of the origins, oriS14, is located within or adjacent to the coding sequence for ribosomal protein S14 on chromosome 2q, and the other, ori-beta, is approximately 17 kbp downstream of the dhfr (dihydrofolic acid reductase) locus on chromosome 2p. The DMI in oriS14 is 127 bp long, and the DMI in ori-beta is 516 bp long. Both DMIs are bilaterally methylated (i.e., all dCs are modified to 5-methyl dC) only in cells that are replicating their DNA. When cell growth and DNA replication are arrested, methylation of CpA, CpT, and CpC dinucleotides is lost and the sequence islands display only a subset of their originally methylated CpG dinucleotides. Several possible roles for DMI-mediated regulation of mammalian chromosomal origins are considered.     

Mapping of the DNA linking tyrosine residue of the PRD1 terminal protein.

DNA replication of PRD1, a lipid-containing phage, is initiated by a protein-priming mechanism. The terminal protein encoded by gene 8 acts as a protein primer in DNA synthesis by forming an initiation complex with the 5'-terminal nucleotide, dGMP. The linkage between the terminal protein and the 5' terminal nucleotide is a tyrosylphosphodiester bond. The PRD1 terminal protein contains 13 tyrosine residues in a total of 259 amino acids. By site-directed mutagenesis of cloned PRD1 gene 8, we replaced 12 of the 13 tyrosine residues in the terminal protein with phenylalanine and the other tyrosine residue with asparagine. Functional analysis of these mutant terminal proteins suggested that tyrosine-190 is the linking amino acid that forms a covalent bond with dGMP. Cyanogen bromide cleavage studies also implicated tyrosine-190 as the DNA-linking amino acid residue of the PRD1 terminal protein. Our results further show that tyrosine residues at both the amino-terminal and the carboxyl-terminal regions are important for the initiation complex forming activity. Predicted secondary structures for the regions around the DNA linking amino acid residues were compared in three terminal proteins (phi 29, adenovirus-2, and PRD1). While the linking amino acids serine-232 (phi 29) and serine-577 (adenovirus-2) are found in beta-turns in hydrophilic regions, the linking tyrosine-190 of the PRD1 terminal protein is found in a beta-sheet in a hydrophobic region.     

Using structural motif templates to identify proteins with DNA binding function

This work describes a method for predicting DNA binding function from structure using 3-dimensional templates. Proteins that bind DNA using small contiguous helix–turn–helix (HTH) motifs comprise a significant number of all DNA-binding proteins. A structural template library of seven HTH motifs has been created from non-homologous DNA-binding proteins in the Protein Data Bank. The templates were used to scan complete protein structures using an algorithm that calculated the root mean squared deviation (rmsd) for the optimal superposition of each template on each structure, based on C_α backbone coordinates. Distributions of rmsd values for known HTH-containing proteins (true hits) and non-HTH proteins (false hits) were calculated. A threshold value of 1.6 Å rmsd was selected that gave a true hit rate of 88.4% and a false positive rate of 0.7%. The false positive rate was further reduced to 0.5% by introducing an accessible surface area threshold value of 990 Ų per HTH motif. The template library and the validated thresholds were used to make predictions for target proteins from a structural genomics project.     

Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria.

An amino acid motif was identified that consists of the sequence HisHydrHisHydrHydrHydr (Hydr--bulky hydrophobic residue) and is conserved in two vast classes of proteins, one of which is involved in initiation and termination of rolling circle DNA replication, or RCR (Rep proteins), and the other in mobilization (conjugal transfer) of plasmid DNA (Mob proteins). Based on analogies with metalloenzymes, it is hypothesized that the two conserved His residues in this motif may be involved in metal ion coordination required for the activity of the Rep and Mob proteins. Rep proteins contained two additional conserved motifs, one of which was located upstream, and the other downstream from the 'two His' motif. The C-terminal motif encompassed the Tyr residue(s) forming the covalent link with nicked DNA. Mob proteins were characterized by the opposite orientation of the conserved motifs, with the (putative) DNA-linking Tyr being located near their N-termini. Both Rep and Mob protein classes further split into several distinct families. Although it was not possible to find a motif or pattern that would be unique for the entire Rep or Mob class, unique patterns were derived for large subsets of the proteins of each class. These observations allowed the prediction of the amino acid residues involved in DNA nicking, which is required for the initiation of RCR or conjugal transfer of single-stranded (ss) DNA, in Rep and Mob proteins encoded by a number of replicons of highly diverse size, structure and origin. It is conjectured that recombination has played a major part in the dissemination of genes encoding related Rep or Mob proteins among the replicons exploiting RCR. It is speculated that the eucaryotic small ssDNA replicons encoding proteins with the conserved RCR motifs and replicating via RCR-related mechanisms, such as geminiviruses and parvoviruses, may have evolved from eubacterial replicons.     

A minimal system for Tn7 transposition: the transposon-encoded proteins TnsA and TnsB can execute DNA breakage and joining reactions that generate circularized Tn7 species

In the presence of ATP and Mg(2+), the bacterial transposon Tn7 translocates via a cut and paste mechanism executed by the transposon-encoded proteins TnsA+TnsB+TnsC+TnsD. We report here that in the presence of Mn(2+), TnsA+TnsB alone can execute the DNA breakage and joining reactions of Tn7 recombination. ATP is not essential in this minimal system, revealing that this cofactor is not directly involved in the chemical steps of recombination. In both the TnsAB and TnsABC+D systems, recombination initiates with double-strand breaks at each transposon end that cut Tn7 away from flanking donor DNA. In the minimal system, breakage occurs predominantly at a single transposon end and the subsequent end-joining reactions are intramolecular, with the exposed 3' termini of a broken transposon end joining near the other end of the Tn7 element in the same donor molecule to form circular transposon species. In contrast, in TnsABC+D recombination, breaks occur at both ends of Tn7 and the two ends join to a target site on a different DNA molecule to form an intermolecular simple insertion. This demonstration of the capacity of TnsAB to execute breakage and joining reactions supports the view that these proteins form the Tn7 transposase.