Facilitated diffusion in chromatin lattices: mechanistic diversity and regulatory potential

The interaction between a protein and a specific DNA site is the molecular basis for vital processes in all organisms. Location of the DNA target site by the protein commonly involves facilitated diffusion. Mechanisms of facilitated diffusion vary among proteins; they include one- and two-dimensional sliding along DNA, direct transfer between uncorrelated sites, as well as combinations of these mechanisms. Facilitated diffusion has almost exclusively been studied in vitro. This review discusses facilitated diffusion in the context of the living cell and proposes a theoretical model for facilitated diffusion in chromatin lattices. Chromatin structure differentially affects proteins in different modes of diffusion. The interplay of facilitated diffusion and chromatin structure can determine the rate of protein association with the target site, the frequency of association-dissociation events at the target site, and, under particular conditions, the occupancy of the target site. Facilitated diffusion is required in vivo for efficient DNA repair and bacteriophage restriction and has potential roles in fine-tuning gene regulatory networks and kinetically compartmentalizing the eukaryotic nucleus.     

Formation, characterization and partial purification of a Tn5 strand transfer complex

DNA transposition reactions typically involve a strand transfer step wherein the transposon ends are covalently joined by the transposase protein to a short target site. There is very little known about the transposase-DNA interactions that direct this process, and thus our overall understanding of the dynamics of DNA transposition reactions is limited. Tn5 presents an attractive system for defining such interactions because it has been possible to solve the structure of at least one Tn5 transposition intermediate: a transpososome formed with pre-cleaved ends. However, insertion specificity in the Tn5 system is low and this has hampered progress in generating target-containing transpososomes that are homogeneous in structure (i.e. where a single target site is engaged) and therefore suitable for biochemical and structural analysis. We have developed a system where the Tn5 transpososome integrates almost exclusively into a single target site within a short DNA fragment. The key to establishing this high degree of insertion specificity was to use a target DNA with tandem repeats of a previously characterized Tn5 insertion hotspot. The target DNA requirements to form this strand transfer complex are evaluated. In addition, we show that target DNAs missing single phosphate groups at specific positions are better substrates for strand transfer complex formation relative to the corresponding unmodified DNA fragments. Moreover, utilization of missing phosphate substrates can increase the degree of target site selection. A method for concentrating and partially purifying the Tn5 strand transfer complex is described.     

Induced bending of plasmid pLS1 DNA by the plasmid-encoded protein RepA

The broad host range streptococcal plasmid pLS1 encodes for a 5.1-kDa repressor protein, RepA. This protein has affinity for DNA (linear or supercoiled) and is translated from the same mRNA as the replication initiator protein RepB. By gel retardation assays, we observed that RepA shows specificity for binding to the plasmid HinfID fragment, which includes the target of the protein. The target of RepA within the plasmid DNA molecule has been located around the plasmid single site ApaLI. This site is included in a region that contains the promoter for the repA and repB genes and is contiguous to the plasmid ori(+). A complex sequence-directed DNA curvature is observed in this region of pLS1. Upon addition of RepA to plasmid linear DNA or to circularly permuted restriction fragments, this intrinsic curvature was greatly enhanced. Thus, a strong RepA-induced bending could be located in the vicinity of the ApaLI site. Visualization of the bent DNA was achieved by electron microscopy of complexes between RepA and plasmid DNA fragments containing the RepA target.     

Target DNA bending is an important specificity determinant in target site selection in Tn10 transposition

The bacterial transposon Tn10 inserts preferentially into specific DNA sequences. DNA footprinting and interference studies have revealed that the Tn10-encoded transposase protein contacts a large stretch of target DNA ( approximately 24 bp) and that the target DNA structure is deformed upon incorporation into the transpososome. Target DNA deformation might contribute significantly to target site selection and thus it is of interest to further define the nature of this deformation. Circular permutation analysis was used to demonstrate that the target DNA is bent upon its incorporation into the transpososome. Two lines of evidence are presented that target DNA bending is an important event in target site selection. First, we demonstrate a correlation between increased target site usage and an increased level of target DNA bending. Second, transposase mutants with relaxed target specificity are shown to cause increased target DNA bending relative to wild-type transposase. This latter observation provides new insight into how relaxed specificity may be achieved. We also show that Ca(2+) facilitates target capture by stabilizing transposase interactions with sequences immediately flanking the insertion site. Ca(2+) could, in theory, exert this effect by stabilizing bends in the target DNA.     

Characterization of recombinant murine leukemia virus integrase.

Retroviral integration involves two DNA substrates that play different roles. The viral DNA substrate is recognized by virtue of specific nucleotide sequences near the end of a double-stranded DNA molecule. The target DNA substrate is recognized at internal sites with little sequence preference; nucleosomal DNA appears to be preferred for this role. Despite this apparent asymmetry in the sequence, structure, and roles of the DNA substrates in the integration reaction, the existence of distinct binding sites for viral and target DNA substrates has been controversial. In this report, we describe the expression in Escherichia coli and purification of Moloney murine leukemia virus integrase as a fusion protein with glutathione S-transferase, characterization of its activity by using several model DNA substrates, and the initial kinetic characterization of its interactions with a model viral DNA substrate. We provide evidence for functionally and kinetically distinct binding sites for viral and target DNA substrates and describe a cross-linking assay for DNA binding at a site whose specificity is consistent with the target DNA binding site.     

Use of Patient-Derived Human Immunodeficiency Virus Type 1 Integrases To Identify a Protein Residue That Affects Target Site Selection

To identify parts of retroviral integrase that interact with cellular DNA, we tested patient-derived human immunodeficiency virus type 1 (HIV-1) integrases for alterations in the choice of nonviral target DNA sites. This strategy took advantage of the genetic diversity of HIV-1, which provided 75 integrase variants that differed by a small number of amino acids. Moreover, our hypothesis that biological pressures on the choice of nonviral sites would be minimal was validated when most of the proteins that catalyzed DNA joining exhibited altered target site preferences. Comparison of the sequences of proteins with the same preferences then guided mutagenesis of a laboratory integrase. The results showed that single amino acid substitutions at one particular residue yielded the same target site patterns as naturally occurring integrases that included these substitutions. Similar results were found with DNA joining reactions conducted with Mn(2+) or with Mg(2+) and were confirmed with a nonspecific alcoholysis assay. Other amino acid changes at this position also affected target site preferences. Thus, this novel approach has identified a residue in the central domain of HIV-1 integrase that interacts with or influences interactions with cellular DNA. The data also support a model in which integrase has distinct sites for viral and cellular DNA.     

Simultaneous recognition of nucleobase and sites of DNA damage: effect of tethered cation on the binding affinity

BACKGROUND: The 3,5-diamino-N-(3-aminopropyl)-6-chloropyrazine-2-carboxamide (DCPC-NH(2)) has been synthesized and characterized by Mass and (1)H NMR. The selective binding of the ligand to thymine (T) target base is investigated by the melting temperature (T(m)) and fluorescence measurements. METHODS: Thermal denaturation study of DNA duplex containing T target base revealed the DeltaT(m) of 5.1 degrees C, while least influence was observed for other target bases. The fluorescence of the ligand DCPC-NH(2) is quenched only upon adding the DNA containing T target base. RESULTS: The binding constant for the interaction of the ligand to T target base containing DNA duplex was determined to be 4.7 (+/-0.3)x10(6) M(-1). The tethered cation in the ligand is found to enhance the binding constant. The ligand binds to both a target nucleotide and an AP site on the complimentary strand for the target strand in a DNA duplex. GENERAL SIGNIFICANCE: Interestingly, the electronic behavior of the ligand depends on the bases flanking the AP site. Its fluorescence is quenched with guanine flanking bases, while it is enhanced with DNA duplex containing T bases flanking an AP site. Finally, the binding modes were visualized by molecular modeling.     

Mutations in endonuclease V that affect both protein-protein association and target site location

A general mechanism by which proteins locate their target sites within large domains of DNA is a one-dimensional facilitated diffusion process in which the protein scans DNA in a nonspecifically bound state. An electrostatic contribution to this type of mechanism has been previously established. This study was designed to question whether other characteristics of a protein's structure might contribute to the scanning mechanism of target site location. In this regard, T4 endonuclease V was shown to establish an ionic strength dependent monomer-dimer equilibrium in solution. A protein dimer interaction site was postulated to exist along a putative alpha-helix containing amino acid residues 54-62. The conservative substitutions of Phe-60----Leu-60 and Phe-59, Phe-60----Leu-59, Leu-60 resulted in mutant enzymes which remained in the monomeric state independent of the ionic strength of the solution. The target site location mechanism of these mutants has also been altered. Under conditions where wild-type endonuclease V processively scans nontarget DNA, the target location mechanism of the monomeric mutant proteins was shifted toward a less processive search. This decrease in the processivity of the mutants was especially surprising because the nontarget DNA binding affinity was found to be significantly increased. Thus, an additional component of the endonuclease V DNA scanning mechanism appears to be the formation of a stable endonuclease V dimer complex.     

A framework for estimating the sensitivity of eDNA surveys

Imperfect sensitivity, or imperfect detection, is a feature of all survey methods that needs to be accounted for when interpreting survey results. Detection of environmental DNA (eDNA) is increasingly being used to infer species distributions, yet the sensitivity of the technique has not been fully evaluated. Sensitivity, or the probability of detecting target DNA given it is present at a site, will depend on both the survey method and the concentration and dispersion of target DNA molecules at a site. We present a model to estimate target DNA concentration and dispersion at survey sites and to estimate the sensitivity of an eDNA survey method. We fitted this model to data from a species‐specific eDNA survey for Oriental weatherloach, Misgurnus anguillicaudatus, at three sites sampled in both autumn and spring. The concentration of target DNA molecules was similar at all three sites in autumn but much higher at two sites in spring. Our analysis showed the survey method had ≥95% sensitivity at sites where target DNA concentrations were ≥11 molecules per litre. We show how these data can be used to compare sampling schemes that differ in the number of field samples collected per site and number of PCR replicates per sample to achieve ≥95% sensitivity at a given target DNA concentration. These models allow researchers to quantify the sensitivity of eDNA survey methods to optimize the probability of detecting target species, and to compare DNA concentrations spatially and temporarily.     

DNA as the Intracellular Secondary Target for Antibacterial Action of Human Neutrophil Peptide-I Against Mycobacterium tuberculosis H37Ra

The secondary intracellular target of human neutrophil peptide-1 has been examined in M. tuberculosis H₃₇Ra. Binding studies with radioiodinated HNP-1 revealed biphasic equilibrium binding kinetics with respect to time. The major site of HNP-1 binding was found to be plasma membrane/cell wall whereas the cytosol appears to be a secondary site. Among the different macromolecules examined, maximum inhibition (75%) was observed in DNA biosynthesis during treatment with HNP-1. The interaction of HNP-1 with mycobacterial genomic DNA on the basis of gel retardation assay revealed HNP-1 binding to DNA. These results indicate that HNP-1 has DNA as the secondary intracellular target for antibacterial action against mycobacteria. Received: 25 October 2000/Accepted: 10 January 2001     

Tn7: a target site-specific transposon

The bacterial transposon Tn7 is an unusual mobile DNA segment. Most transposable elements move at low-frequency and display little target site-selectivity. By contrast, Tn7 inserts at high-frequency into a single specific site in the chromosomes of many bacteria. In the absence of this specific site, called attTn7 in Escherichia coli where Tn7 has been most extensively studied, Tn7 transposes at low-frequency and inserts into many different sites. Much has recently been learned about Tn7 transposition from both genetic and biochemical studies. The Tn7 recombination machinery is elaborate and includes a large number of Tn7-encoded proteins, probably host-encoded proteins and also rather large cis-acting transposition sequences at the transposon termini and at the target site. Dissection of the Tn7 transposition mechanism has revealed that the DNA strand breakage and joining reactions that underlie the translocation of Tn7 have several unusual features.     

Transposition of a long member of the L1 major interspersed DNA family into the mouse beta globin gene locus.

A long member of the highly repeated long interspersed DNA family L1Md (for L1 in Mus domesticus) has integrated by transposition into a target site which lies between the two adult beta globin genes of mouse. DNA hybridization and nucleotide sequence analysis show that this target site, which is part of the single copy DNA flanking the globin genes, is interrupted by the L1 element in one chromosome but is uninterrupted in both allelic and ancestral chromosomes. Other large DNA rearrangements of the region between the two adult beta globin genes are also associated with these allelic chromosomes, and include insertions or deletions of both single copy DNA and simple and complex repetitive DNA. This has caused extensive reorganization of this intergenic region. However, the distance between the two genes flanking this region remains conserved, suggesting that the spacing of the globin genes may be subject to conservative selection.     

Tn7 transposition as a probe of cis interactions between widely separated (190 kilobases apart) DNA sites in the Escherichia coli chromosome.

We have used the bacterial transposon Tn7 to examine communication between widely separated DNA sites in the Escherichia coli chromosome. Using Tn7 target immunity, a regulatory feature of transposition which influences target selection, we have evaluated (i) how the presence of Tn7 sequences at one DNA site affects Tn7 insertion into another site in the same DNA molecule and (ii) the nucleotide distances over which the two sites are able to communicate. We demonstrate that Tn7 sequences at one chromosomal site act at a distance to inhibit insertion of Tn7 elsewhere in that DNA as far away as 190 kb, reflecting effective long-range cis interactions. We have found that while target immunity is effective over a substantial region of the chromosome, insertion of Tn7 into a more distant site 1.9 Mb away in the same DNA is not inhibited; this observation provides evidence that target immunity relies on DNA spacing. We also find that within the region of the chromosome affected by target immunity, the magnitude of the immune effect is greater at close DNA sites than DNA sites farther away, suggesting that target immunity is distance dependent. We also extend the characterization of the Tn7 end-sequences involved in transposition and target immunity and describe how Tn7 target immunity can be used as a tool for probing bacterial chromosome structure.     

Heterodimeric DNA methyltransferases as a platform for creating designer zinc finger methyltransferases for targeted DNA methylation in cells

The ability to target methylation to specific genomic sites would further the study of DNA methylation’s biological role and potentially offer a tool for silencing gene expression and for treating diseases involving abnormal hypomethylation. The end-to-end fusion of DNA methyltransferases to zinc fingers has been shown to bias methylation to desired regions. However, the strategy is inherently limited because the methyltransferase domain remains active regardless of whether the zinc finger domain is bound at its cognate site and can methylate non-target sites. We demonstrate an alternative strategy in which fragments of a DNA methyltransferase, compromised in their ability to methylate DNA, are fused to two zinc fingers designed to bind 9 bp sites flanking a methylation target site. Using the naturally heterodimeric DNA methyltransferase M.EcoHK31I, which methylates the inner cytosine of 5′-YGGCCR-3′, we demonstrate that this strategy can yield a methyltransferase capable of significant levels of methylation at the target site with undetectable levels of methylation at non-target sites in Escherichia coli. However, some non-target methylation could be detected at higher expression levels of the zinc finger methyltransferase indicating that further improvements will be necessary to attain the desired exclusive target specificity.     

DNA rearrangement associated with the integration of T-DNA in tobacco: an example for multiple duplications of DNA around the integration target

Transferred DNA (T-DNA) of the tumor-inducing (Ti) plasmid is transferred from Agrobacterium tumefaciens to plant cells and is stably integrated into the plant nuclear genome. By the inverse polymerase chain reaction DNA fragments were amplified that contained the T-DNA/plant DNA junctions from the total DNA of a transgenic tobacco plant that had a single copy of the T-DNA in a repetitive region of its genome. A DNA fragment containing the target site was amplified from the total DNA of non-transformed tobacco by the polymerase chain reaction using high-stringency conditions. Comparison of the nucleotide sequence of the target site with those of the T-DNA/plant DNA junctions revealed that various duplications of short stretches of nucleotide sequences around the target and in the incoming T-DNA had accompanied the integration of the T-DNA. A deletion of 16 bp at the target site was also found and the target site was similar, in terms of nucleotide sequence, to regions around the breakpoints of the T-DNA. This finding provides a clear example of the occurrence of complex rearrangements during the integration of T-DNA.     

The reaction mechanism of FokI excludes the possibility of targeting zinc finger nucleases to unique DNA sites

The FokI endonuclease is a monomeric protein with discrete DNA-recognition and catalytic domains. The latter has only one active site so, to cut both strands, the catalytic domains from two monomers associate to form a dimer. The dimer involving a monomer at the recognition site and another from free solution is less stable than that from two proteins tethered to the same DNA. FokI thus cleaves DNA with two sites better than one-site DNA. The two sites can be immediately adjacent, but they can alternatively be many hundreds of base pairs apart, in either inverted or repeated orientations. The catalytic domain of FokI is often a component of zinc finger nucleases. Typically, the zinc finger domains of two such nucleases are designed to recognize two neighbouring DNA sequences, with the objective of cutting the DNA exclusively between the target sequences. However, this strategy fails to take account of the fact that the catalytic domains of FokI can dimerize across distant sites or even at a solitary site. Additional copies of either target sequence elsewhere in the chromosome must elicit off-target cleavages.     

Altered target site specificity variants of the I-PpoI His-Cys box homing endonuclease

We used a yeast one-hybrid assay to isolate and characterize variants of the eukaryotic homing endonuclease I-PpoI that were able to bind a mutant, cleavage-resistant I-PpoI target or ‘homing’ site DNA in vivo. Native I-PpoI recognizes and cleaves a semi-palindromic 15-bp target site with high specificity in vivo and in vitro. This target site is present in the 28S or equivalent large subunit rDNA genes of all eukaryotes. I-PpoI variants able to bind mutant target site DNA had from 1 to 8 amino acid substitutions in the DNA–protein interface. Biochemical characterization of these proteins revealed a wide range of site–binding affinities and site discrimination. One-third of variants were able to cleave target site DNA, but there was no systematic relationship between site-binding affinity and site cleavage. Computational modeling of several variants provided mechanistic insight into how amino acid substitutions that contact, or are adjacent to, specific target site DNA base pairs determine I-PpoI site-binding affinity and site discrimination, and may affect cleavage efficiency.     

Kinetics of target site localization of a protein on DNA: a stochastic approach

It is widely recognized that the cleaving rate of a restriction enzyme on target DNA sequences is several orders-of-magnitude faster than the maximal one calculated from the diffusion-limited theory. It was therefore commonly assumed that the target site interaction of a restriction enzyme with DNA has to occur via two steps: one-dimensional diffusion along a DNA segment, and long-range jumps coming from association-dissociation events. We propose here a stochastic model for this reaction which comprises a series of one-dimensional diffusions of a restriction enzyme on nonspecific DNA sequences interrupted by three-dimensional excursions in the solution until the target sequence is reached. This model provides an optimal finding strategy which explains the fast association rate. Modeling the excursions by uncorrelated random jumps, we recover the expression of the mean time required for target site association to occur given by Berg et al. in 1981, and we explicitly give several physical quantities describing the stochastic pathway of the enzyme. For competitive target sites we calculate two quantities: processivity and preference. By comparing these theoretical expressions to recent experimental data obtained for EcoRV-DNA interaction, we quantify: 1), the mean residence time per binding event of EcoRV on DNA for a representative one-dimensional diffusion coefficient; 2), the average lengths of DNA scanned during the one-dimensional diffusion (during one binding event and during the overall process); and 3), the mean time and the mean number of visits needed to go from one target site to the other. Further, we evaluate the dynamics of DNA cleavage with regard to the probability for the restriction enzyme to perform another one-dimensional diffusion on the same DNA substrate following a three-dimensional excursion.     

Ordered DNA release and target capture in RAG transposition

Following V(D)J cleavage, the newly liberated DNA signal ends can be either fused together into a signal joint or used as donor DNA in RAG-mediated transposition. We find that both V(D)J cleavage and release of flanking coding DNA occur before the target capture step of transposition can proceed; no coding DNA is ever detected in the target capture complex. Separately from its role in V(D)J cleavage, the DDE motif of the RAG1/2 active site is specifically required for target DNA capture. The requirement for cleavage and release of coding DNA prior to either physical target binding or functional target commitment suggests that the RAG1/2 transposase contains a single binding site for non-RSS DNA that can accommodate either target DNA or coding DNA, but not both together. Perhaps the presence of coding DNA may aid in preventing transpositional resolution of V(D)J recombination intermediates.