Allosteric interactions of DNA and nucleotides with S. cerevisiae RSC

RSC (remodel the structure of chromatin) is an essential chromatin remodeler of Saccharomyces cerevisiae that has been shown to have DNA translocase properties. We studied the DNA binding properties of a "trimeric minimal RSC" (RSCt) of the RSC chromatin remodeling complex and the effect of nucleotides on this interaction using fluorescence anisotropy. RSCt binds to 20 bp fluorescein-labeled double-stranded DNA with a K(d) of ～100 nM. The affinity of RSCt for DNA is reduced in the presence of AMP-PNP and ADP in a concentration-dependent manner with the addition of AMP-PNP having more pronounced effect. These differences in the magnitude at which the binding of ADP and AMP-PNP affects the affinity of DNA binding by RSCt suggest that the physical movement of the enzyme along DNA begins between the binding of ATP and its subsequent hydrolysis. Furthermore, the fact that the highest affinity for DNA binding by RSCt occurs in the absence of bound nucleotide offers a mechanistic explanation for the apparent low processivity of DNA translocation by the enzyme.     

Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme.

A fluorescence assay was used to measure the processivity of Escherichia coli recBCD enzyme helicase activity. Under standard conditions, recBCD enzyme unwinds an average of 30 +/- 3.2 kilobase pairs (kb)/DNA end before dissociating. The average processivity (P obs) of DNA unwinding under these conditions is 0.99997, indicating that the probability of unwinding another base pair is 30,000-fold greater than the probability of dissociating from the double-stranded DNA. The average number of base pairs unwound per binding event (N) is sensitive to both mono- and divalent salt concentration and ranges from 36 kb at 80 mM NaCl to 15 kb at 280 mM NaCl. The processivity of unwinding increases in a hyperbolic manner with increasing ATP concentration, yielding a KN value for ATP of 41 +/- 9 microM and a limiting value of 32 +/- 1.8 kb/end for the number of base pairs unwound. The importance of the processivity of recBCD enzyme helicase activity to the recBCD enzyme-dependent stimulation of recombination at Chi sites observed in vivo is discussed.     

Multiple Aspects of ATP-Dependent Nucleosome Translocation by RSC and Mi-2 Are Directed by the Underlying DNA Sequence

Background

Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the nucleosome positions can be predicted simply by DNA sequence, especially within promoter regions. This seemingly contrasts with remodeler induced nucleosome mobility. The ability of remodeling enzymes to mobilise nucleosomes over short DNA distances is well documented. However, the nucleosome translocation processivity along DNA remains elusive. Furthermore, it is unknown what determines the initial direction of movement and how new nucleosome positions are adopted.

Methodology/Principal Findings

We have used AFM imaging and high resolution PAGE of mononucleosomes on 600 and 2500 bp DNA molecules to analyze ATP-dependent nucleosome repositioning by native and recombinant SNF2-type enzymes. We report that the underlying DNA sequence can control the initial direction of translocation, translocation distance, as well as the new positions adopted by nucleosomes upon enzymatic mobilization. Within a strong nucleosomal positioning sequence both recombinant Drosophila Mi-2 (CHD-type) and native RSC from yeast (SWI/SNF-type) repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalyzed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps.

Conclusions/Significance

Nucleosome repositioning thus appears to be influenced by both remodeler intrinsic and DNA sequence specific properties that interplay to define ATPase-catalyzed repositioning. Here we propose a successive three-step framework consisting of initiation, translocation and release steps to describe SNF2-type enzyme mediated nucleosome translocation along DNA. This conceptual framework helps resolve the apparent paradox between the high abundance of ATP-dependent remodelers per nucleus and the relative success of sequence-based predictions of nucleosome positioning in vivo.     

Chromatin remodeling through directional DNA translocation from an internal nucleosomal site

The RSC chromatin remodeler contains Sth1, an ATP-dependent DNA translocase. On DNA substrates, RSC/Sth1 tracks along one strand of the duplex with a 3' --> 5' polarity and a tracking requirement of one base, properties that may enable directional DNA translocation on nucleosomes. The binding of RSC or Sth1 elicits a DNase I-hypersensitive site approximately two DNA turns from the nucleosomal dyad, and the binding of Sth1 requires intact DNA at this location. Results with various nucleosome substrates suggest that RSC/Sth1 remains at a fixed position on the histone octamer and that Sth1 conducts directional DNA translocation from a location about two turns from the nucleosomal dyad, drawing in DNA from one side of the nucleosome and pumping it toward the other. These studies suggest that nucleosome mobilization involves directional DNA translocation initiating from a fixed internal site on the nucleosome.     

Processive translocation mechanism of the human Bloom’s syndrome helicase along single-stranded DNA

BLM, one of the human RecQ helicases, plays a fundamental role in homologous recombination-based error-free DNA repair pathways, which require its translocation and DNA unwinding activities. Although translocation is essential in vivo during DNA repair processes and it provides a framework for more complex activities of helicases, including strand separation and nucleoprotein displacement, its mechanism has not been resolved for any human DNA helicase. Here, we present a quantitative model for the translocation of a monomeric form of BLM along ssDNA. We show that BLM performs translocation at a low adenosine triphosphate (ATP) coupling ratio (1 ATP consumed/1 nucleotide traveled) and moderate processivity (with a mean number of 50 nucleotides traveled in a single run). We also show that the rate-limiting step of the translocation cycle is a transition between two ADP-bound enzyme states. Via opening of the helicase core, this structural change may drive the stepping of BLM along the DNA track by a directed inchworm mechanism. The data also support the conclusion that BLM performs double-stranded DNA unwinding by fully active duplex destabilization.     

ATP-dependent translocation of proteins along single-stranded DNA: models and methods of analysis of pre-steady state kinetics

Processive DNA helicases are able to translocate along single-stranded DNA (ssDNA) with biased directionality in a nucleoside triphosphate-dependent reaction, although translocation is not generally sufficient for helicase activity. An understanding of the mechanism of protein translocation along ssDNA requires pre-steady state transient kinetic experiments. Although ensemble experimental approaches have been developed recently for the study of translocation of proteins along DNA, quantitative analysis of the complete time-courses from these experiments, which is needed to obtain quantitative estimates of translocation kinetic parameters (rate constants, processivity, step sizes and ATP coupling) has been lacking. We discuss three ensemble transient kinetic experiments that can be used to study protein translocation along ssDNA, along with the advantages and limitations of each approach. We further describe methods to analyze the complete kinetic time-courses obtained from such experiments performed with a series of ssDNA lengths under "single-round" conditions (i.e. in the absence of re-binding of dissociated protein to DNA). These analysis methods utilize a sequential "n-step" model for protein translocation along ssDNA and enable quantitative determinations of the rate constant, processivity and step size for translocation through global non-linear least-squares fitting of the full time-courses.     

DNA Structure Specificity Conferred on a Replicative Helicase by Its Loader

Prokaryotic and eukaryotic replicative helicases can translocate along single-stranded and double-stranded DNA, with the central cavity of these multimeric ring helicases being able to accommodate both forms of DNA. Translocation by such helicases along single-stranded DNA results in the unwinding of forked DNA by steric exclusion and appears critical in unwinding of parental strands at the replication fork, whereas translocation over double-stranded DNA has no well-defined role. We have found that the accessory factor, DnaC, that promotes loading of the Escherichia coli replicative helicase DnaB onto single-stranded DNA may also act to confer DNA structure specificity on DnaB helicase. When present in excess, DnaC inhibits DnaB translocation over double-stranded DNA but not over single-stranded DNA. Inhibition of DnaB translocation over double-stranded DNA requires the ATP-bound form of DnaC, and this inhibition is relieved during translocation over single-stranded DNA indicating that stimulation of DnaC ATPase is responsible for this DNA structure specificity. These findings demonstrate that DnaC may provide the DNA structure specificity lacking in DnaB, limiting DnaB translocation to bona fide replication forks. The ability of other replicative helicases to translocate along single-stranded and double-stranded DNA raises the possibility that analogous regulatory mechanisms exist in other organisms.     

Mechanism of ATP-dependent translocation of E.coli UvrD monomers along single-stranded DNA

Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. Using stopped-flow methods we have examined the kinetic mechanism of translocation of UvrD monomers along single-stranded DNA (ssDNA) in vitro by monitoring the transient kinetics of arrival of protein at the 5'-end of the ssDNA. Arrival at the 5'-end was monitored by the effect of protein on the fluorescence intensity of fluorophores (Cy3 or fluorescein) attached to the 5'-end of a series of oligodeoxythymidylates varying in length from 16 to 124 nt. We find that UvrD monomers are capable of ATP-dependent translocation along ssDNA with a biased 3' to 5' directionality. Global non-linear least-squares analysis of the full kinetic time-courses in the presence of a protein trap to prevent rebinding of free protein to the DNA using the methods described in the accompanying paper enabled us to obtain quantitative estimates of the kinetic parameters for translocation. We find that UvrD monomers translocate in discrete steps with an average kinetic step-size, m=3.68(+/-0.03) nt step(-1), a translocation rate constant, kt=51.3(+/-0.6) steps s(-1), (macroscopic translocation rate, mkt=189.0(+/-0.7) nt s(-1)), with a processivity corresponding to an average translocation distance of 2400(+/-600) nt before dissociation (10 mM Tris-HCl (pH 8.3), 20 mM NaCl, 20% (v/v) glycerol, 25 degrees C). However, in spite of its ability to translocate rapidly and efficiently along ssDNA, a UvrD monomer is unable to unwind even an 18 bp duplex in vitro. DNA helicase activity in vitro requires a UvrD dimer that unwinds DNA with a similar kinetic step-size of 4-5 bp step(-1), but an approximately threefold slower unwinding rate of 68(+/-9) bp s(-1) under the same solution conditions, indicating that DNA unwinding activity requires more than the ability to simply translocate directionally along ss-DNA.     

Human mitochondrial DNA polymerase holoenzyme: reconstitution and characterization

We have reconstituted the holoenzyme of the human mitochondrial DNA polymerase from cloned and overexpressed catalytic and accessory subunits. We have examined the polymerization activity of the catalytic subunit alone and of the holoenzyme to establish the function of the accessory subunit in this two subunit enzyme. The accessory subunit associates with the catalytic subunit with a dissociation constant of 35 +/- 16 nM as measured by the concentration dependence of its effect in stimulating maximal DNA binding and polymerization. At saturating concentrations, the accessory subunit contributes to every kinetic parameter examined to facilitate tighter binding of DNA and nucleotide and faster replication. The accessory protein makes the DNA binding 3.5-fold tighter (K(d) of 9.9 +/- 2.1 nM compared to 39 +/- 10 nM for the catalytic subunit alone) without significantly affecting the DNA dissociation rate (0.02 +/- 0.001 compared to 0.03 +/- 0.001 s(-)(1)). The ground-state nucleotide binding is improved from 4.7 +/- 2.0 to 0.78 +/- 0.065 microM, and the maximum DNA polymerization rate is increased from 8.7 +/- 1.1 to 45 +/- 1 s(-)(1) by the addition of the accessory protein. This leads to an increase in processivity from an estimated 290 +/- 46 to 2250 +/- 162. Although the accessory protein has been described as a "processivity factor" because of its effect on the ratio of rate constants defining processivity, this terminology falls short of adequately describing the profound effects of the small subunit on nucleotide-binding and incorporation catalyzed by the large subunit. By using the complete holoenzyme, we can now proceed with a comprehensive analysis of the structural and mechanistic determinants of enzyme specificity that govern toxicity of nucleoside analogues used in the treatment of viral infections such as AIDS.     

T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA

DNA helicases are molecular motors that use the energy from NTP hydrolysis to drive the process of duplex DNA strand separation. Here, we measure the translocation and energy coupling efficiency of a replicative DNA helicase from bacteriophage T7 that is a member of a class of helicases that assembles into ring-shaped hexamers. Presteady state kinetics of DNA-stimulated dTTP hydrolysis activity of T7 helicase were measured using a real time assay as a function of ssDNA length, which provided evidence for unidirectional translocation of T7 helicase along ssDNA. Global fitting of the kinetic data provided an average translocation rate of 132 bases per second per hexamer at 18 degrees C. While translocating along ssDNA, T7 helicase hydrolyzes dTTP at a rate of 49 dTTP per second per hexamer, which indicates that the energy from hydrolysis of one dTTP drives unidirectional movement of T7 helicase along two to three bases of ssDNA. One of the features that distinguishes this ring helicase is its processivity, which was determined to be 0.99996, which indicated that T7 helicase travels on an average about 75kb of ssDNA before dissociating. We propose that the ability of T7 helicase to translocate unidirectionally along ssDNA in an efficient manner plays a crucial role in DNA unwinding.     

Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase

DNA methyltransferase Dnmt1 ensures clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during replication. Dnmt1 is targeted to replication foci, interacts with PCNA, and favors methylating the hemimethylated form of CpG sites. To understand the underlying mechanism of its maintenance function, we purified recombinant forms of full-length Dnmt1, a truncated form of Dnmt1-(291-1620) lacking the binding sites for PCNA and DNA and examined their processivity using a series of long unmethylated and hemimethylated DNA substrates. Direct analysis of methylation patterns using bisulfite-sequencing and hairpin-PCR techniques demonstrated that full-length Dnmt1 methylates hemimethylated DNA with high processivity and a fidelity of over 95%, but unmethylated DNA with much less processivity. The truncated form of Dnmt1 showed identical properties to full-length Dnmt1 indicating that the N-terminal 290-amino acid residue region of Dnmt1 is not required for preferential activity toward hemimethylated sites or for processivity of the enzyme. Remarkably, our analyses also revealed that Dnmt1 methylates hemimethylated CpG sites on one strand of double-stranded DNA during a single processive run. Our findings suggest that these inherent enzymatic properties of Dnmt1 play an essential role in the faithful and efficient maintenance of methylation patterns in the mammalian genome.     

The RSC chromatin remodelling ATPase translocates DNA with high force and small step size

ATP-dependent chromatin remodelling complexes use the energy of ATP hydrolysis to reposition and reconfigure nucleosomes. Despite their diverse functions, all remodellers share highly conserved ATPase domains, many shown to translocate DNA. Understanding remodelling requires biophysical knowledge of the DNA translocation process: how the ATPase moves DNA and generates force, and how translocation and force generation are coupled on nucleosomes. Here, we characterize the real-time activity of a minimal RSC translocase 'motor' on bare DNA, using high-resolution optical tweezers and a 'tethered' translocase system. We observe on dsDNA a processivity of ～35 bp, a speed of ～25 bp/s, and a step size of 2.0 (±0.4, s.e.m.) bp. Surprisingly, the motor is capable of moving against high force, up to 30 pN, making it one of the most force-resistant motors known. We also provide evidence for DNA 'buckling' at initiation. These observations reveal the ATPase as a powerful DNA translocating motor capable of disrupting DNA-histone interactions by mechanical force.     

Bipolar DNA translocation contributes to highly processive DNA unwinding by RecBCD enzyme

We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stopped-flow dye displacement assay for unwinding activity, we test this idea by analyzing mutant RecBCD enzymes in which either of the two helicase motors is inactivated by mutagenesis. Like the wild-type RecBCD enzyme, the two mutant proteins maintain the ability to bind tightly to blunt duplex DNA ends in the absence of ATP. However, the rate of forward translocation for the RecB motor-defective enzyme is only approximately 30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is approximately 50%. More significantly, the processivity of translocation is substantially reduced by approximately 25- and 6-fold for each mutant enzyme, respectively. Despite retaining the capacity to bind blunt dsDNA, the RecB-mutant enzyme has lost the ability to unwind DNA unless the substrate contains a short 5'-terminated single-stranded DNA overhang. The consequences of this observation for the architecture of the single-stranded DNA motors in the initiation complex are discussed.     

Proliferating cell nuclear antigen loaded onto double-stranded DNA: dynamics, minor groove interactions and functional implications

Proliferating cell nuclear antigen (PCNA) acts as a biologically essential processivity factor that encircles DNA and provides binding sites for polymerase, flap endonuclease-1 (FEN-1) and ligase during DNA replication and repair. We have computationally characterized the interactions of human and Archaeoglobus fulgidus PCNA trimer with double-stranded DNA (ds DNA) using multi-nanosecond classical molecular dynamics simulations. The results reveal the interactions of DNA passing through the PCNA trimeric ring including the contacts formed, overall orientation and motion with respect to the sliding clamp. Notably, we observe pronounced tilting of the axis of dsDNA with respect to the PCNA ring plane reflecting interactions between the DNA phosphodiester backbone and positively charged arginine and lysine residues lining the PCNA inner surface. Covariance matrix analysis revealed a pattern of correlated motions within and between the three equivalent subunits involving the PCNA C-terminal region and linker strand associated with partner protein binding sites. Additionally, principal component analysis identified low frequency global PCNA subunit motions suitable for translocation along duplex DNA. The PCNA motions and interactions with the DNA minor groove, identified here computationally, provide an unexpected basis for PCNA to act in the coordinated handoff of intermediates from polymerase to FEN-1 to ligase during DNA replication and repair.     

Single molecule imaging of Tid1/Rdh54, a Rad54 homolog that translocates on duplex DNA and can disrupt joint molecules

The Saccharomyces cerevisiae Tid1 protein is important for the recombinational repair of double-stranded DNA breaks during meiosis. Tid1 is a member of Swi2/Snf2 family of chromatin remodeling proteins and shares homology with Rad54. Members of this family hydrolyze ATP and promote 1) chromatin remodeling, 2) DNA topology alterations, and 3) displacement of proteins from DNA. All of these activities are presumed to require translocation of the protein on DNA. Here we use single-molecule visualization to provide direct evidence for the ability of Tid1 to translocate on DNA. Tid1 translocation is ATP-dependent, and the velocities are broadly distributed, with the average being 84 +/- 39 base pairs/s. Translocation is processive, with the average molecule traveling approximately 10,000 base pairs before pausing or dissociating. Many molecules display simple monotonic unidirectional translocation, but the majority display complex translocation behavior comprising intermittent pauses, direction reversals, and velocity changes. Finally, we demonstrate that translocation by Tid1 on DNA can result in disruption of three-stranded DNA structures. The ability of Tid1 translocation to clear DNA of proteins and to migrate recombination intermediates may be of critical importance for DNA repair and chromosome dynamics.     

Mutations Altering a Structurally Conserved Loop-Helix-Loop Region of a Viral Packaging Motor Change DNA Translocation Velocity and Processivity

Many double-stranded DNA viruses employ ATP-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. Here, we show via direct single-molecule measurements that a mutation T194M downstream of the Walker B motif in the phage λ gpA packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation G212S in the putative C (coupling) motif causes a 3-fold reduction in velocity and a 6-fold reduction in processivity. Meanwhile a T194M pseudorevertant (T194V) showed a near restoration of the wild-type dynamics. Structural comparisons and modeling show that these mutations are in a loop-helix-loop region that positions the key residues of the catalytic motifs, Walker B and C, in the ATPase center and is structurally homologous with analogous regions in chromosome transporters and SF2 RNA helicases. Together with recently published studies of SpoIIIE chromosome transporter and Ded1 RNA helicase mutants, these findings suggest the presence of a structurally conserved region that may be a part of the mechanism that determines motor velocity and processivity in several different types of nucleic acid translocases.     

Processivity of nucleic acid unwinding and translocation by helicases

Helicases are a class of enzymes that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid (NA) strand separation. Besides the NA unwinding speed, another important factor for the helicase activity is the NA unwinding processivity. Here, we study the NA unwinding processivity with an analytical model that captures the phenomenology of the NA unwinding process. First, we study the processivity of the non‐hexameric helicase that can unwind NA efficiently in the form of a monomer and the processivity of the hexameric helicase that can unwind DNA effectively, providing quantitative explanations of the available single‐molecule experimental data. Then, we study the processivity of the non‐hexameric helicases, in particular UvrD, in the form of a dimer and compare with that in the form of a monomer. The available single‐molecule and some biochemical data showing that while UvrD monomer is a highly processive single‐stranded DNA translocase it is inactive in DNA unwinding, whereas other biochemical data showing that UvrD is active in both single‐stranded DNA translocation and DNA unwinding in the form of a monomer can be explained quantitatively and consistently. In addition, the recent single‐molecule data are also explained quantitatively showing that constraining the 2B subdomain in closed conformation by intramolecular cross‐linking can convert Rep monomer with a very poor DNA unwinding activity into a superhelicase that can unwind more than thousands of DNA base pairs processively, even against a large opposing force. Proteins 2016; 84:1590–1605. © 2016 Wiley Periodicals, Inc.     

Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel

T7 Exonuclease (T7 Exo) DNA digestion reactions were studied using direct single-molecule observations in microflow channels. DNA digestion reactions were directly observed by staining template DNA double-stranded regions with SYTOX Orange and staining single-stranded (digested) regions with a fluorescently labeled ssDNA-recognizing peptide (ssBP-488). Sequentially acquired photographs demonstrated that a double-stranded region monotonously shortened as a single-stranded region monotonously increased from the free end during a DNA digestion reaction. Furthermore, DNA digestion reactions were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule, with an estimated average DNA digestion rate of 5.7 bases/s and a processivity of 6692 bases. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses. The average DNA digestion rate was estimated to be 5.3 bases/s with a processivity of 5072 bases. Thus, the use of our direct single-molecule observations using a fluorescently labeled ssDNA-recognizing peptide (ssBP-488) was an effective analytic method for investigating DNA metabolic processes.     

Real-time observation of DNA translocation by the type I restriction modification enzyme EcoR124I

Type I restriction enzymes bind sequence-specifically to unmodified DNA and subsequently pull the adjacent DNA toward themselves. Cleavage then occurs remotely from the recognition site. The mechanism by which these members of the superfamily 2 (SF2) of helicases translocate DNA is largely unknown. We report the first single-molecule study of DNA translocation by the type I restriction enzyme EcoR124I. Mechanochemical parameters such as the translocation rate and processivity, and their dependence on force and ATP concentration, are presented. We show that the two motor subunits of EcoR124I work independently. By using torsionally constrained DNA molecules, we found that the enzyme tracks along the helical pitch of the DNA molecule. This assay may be directly applicable to investigating the tracking of other DNA-translocating motors along their DNA templates.     

Nanopore sensor for fast label-free detection of short double-stranded DNAs

Functionalizing surface enhanced the molecular sensing ability of a fabricated nanopore by increasing the translocation duration time for a short double-stranded DNA. The surface of nanopore was derivatized with γ-aminopropyltriethoxysilane and the positively charged surface attracted DNA molecules when they were in the vicinity of nanopore. The translocation duration time of DNA increased due to the strong electrostatic interaction and it enabled us to detect a short double-stranded DNA (<1 kbp) that is under the size limit of a conventional solid state nanopore sensor. Both 539 and 910 bp double-stranded DNAs were analyzed with the surface functionalized nanopore and their translocation kinetics are presented in this work. The new feature of the surface modified nanopore that can detect short double-stranded DNA molecules could readily be applied for a rapid label-free diagnostic analysis in a Lab-On-a-Chip type DNA sensor.