Biomedical subjects

Mark D Szczelkun

Publications and source records attributed to Mark D Szczelkun.

12 recordsLinked to original sources

PubMed2006

Direct and random routing of a molecular motor protein at a DNA junction.

With the aim of investigating how motor proteins negotiate DNA nanostructures, we produced test circuits based on recombination intermediates in which 1D translocation across a Holliday junction (HJ) could be assessed by subsequent triplex displacement signals on each DNA arm. Using the EcoR124I restriction-modification enzyme, a 3'-5' double-strand DNA (dsDNA) translocase, we could show that the motor will tend to follow its translocated strand across a junction. Nonetheless, as the frequency of junction bypass events increases, the motor will occasionally jump tracks.

DNA, Cruciform↗

PubMed2006

When a helicase is not a helicase: dsDNA tracking by the motor protein EcoR124I.

Using a combination of single molecule and bulk solution measurements, we have examined the DNA translocation activity of a helicase, the Type I restriction modification enzyme EcoR124I. We find that EcoR124I can translocate past covalent interstrand crosslinks, inconsistent with an obligatory unwinding mechanism. Instead, translocation of the intact dsDNA occurs principally via contacts to the sugar-phosphate backbone and bases of the 3'-5' strand; contacts to the 5'-3' strand are not essential for motion but do play a key role in stabilising the motor on the DNA. A model for dsDNA translocation is presented that could be applicable to a wide range of other enzyme complexes that are also labelled as helicases but which do not have actual unwinding activity.

DNA↗

PubMed2005

Dynamics of initiation, termination and reinitiation of DNA translocation by the motor protein EcoR124I.

Type I restriction enzymes use two motors to translocate DNA before carrying out DNA cleavage. The motor function is accomplished by amino-acid motifs typical for superfamily 2 helicases, although DNA unwinding is not observed. Using a combination of extensive single-molecule magnetic tweezers and stopped-flow bulk measurements, we fully characterized the (re)initiation of DNA translocation by EcoR124I. We found that the methyltransferase core unit of the enzyme loads the motor subunits onto adjacent DNA by allowing them to bind and initiate translocation. Termination of translocation occurs owing to dissociation of the motors from the core unit. Reinitiation of translocation requires binding of new motors from solution. The identification and quantification of further initiation steps--ATP binding and extrusion of an initial DNA loop--allowed us to deduce a complete kinetic reinitiation scheme. The dissociation/reassociation of motors during translocation allows dynamic control of the restriction process by the availability of motors. Direct evidence that this control mechanism is relevant in vivo is provided.

Adenosine Triphosphate↗

PubMed2005

Characterization of the Type III restriction endonuclease PstII from Providencia stuartii.

A new Type III restriction endonuclease designated PstII has been purified from Providencia stuartii. PstII recognizes the hexanucleotide sequence 5'-CTGATG(N)(25-26/27-28)-3'. Endonuclease activity requires a substrate with two copies of the recognition site in head-to-head repeat and is dependent on a low level of ATP hydrolysis ( approximately 40 ATP/site/min). Cleavage occurs at just one of the two sites and results in a staggered cut 25-26 nt downstream of the top strand sequence to generate a two base 5'-protruding end. Methylation of the site occurs on one strand only at the first adenine of 5'-CATCAG-3'. Therefore, PstII has characteristic Type III restriction enzyme activity as exemplified by EcoPI or EcoP15I. Moreover, sequence asymmetry of the PstII recognition site in the T7 genome acts as an historical imprint of Type III restriction activity in vivo. In contrast to other Type I and III enzymes, PstII has a more relaxed nucleotide specificity and can cut DNA with GTP and CTP (but not UTP). We also demonstrate that PstII and EcoP15I cannot interact and cleave a DNA substrate suggesting that Type III enzymes must make specific protein-protein contacts to activate endonuclease activity.

Amino Acid Sequence↗

PubMed2005

Subunit assembly modulates the activities of the Type III restriction-modification enzyme PstII in vitro.

We demonstrate that, like other Type III restriction endonuclease, PstII does not turnover such that a DNA substrate is only fully cleaved at a Res2Mod2-to-site ratio of approximately 1:1. However, unlike other Type III enzymes, the cleavage rate profiles varied with protein concentration: using 5 nM DNA and 25 nM PstII, approximately half of the DNA was cut at a fast rate while the remainder was cut 24 times more slowly; in comparison, with 100 nM PstII cleavage occurs at a single fast rate. The inclusion of the methyl donor S-adenosyl methionine does not alter the rates with 100 nM PstII but with 25 nM PstII the reaction stopped after completion of the initial fast cleavage phase owing to methylation. Concentration-dependent rates were also observed in methylation assays: at 100 nM PstII, a single slow rate was measured while at lower PstII concentrations both fast and slow rates were measured. We propose a model in which the intact Res2Mod2 complex favoured at high PstII concentrations is a fast endonuclease/slow methyltransferase while the various subassemblies which coexist at lower concentrations are fast methyltransferases. A potential role for disassembly in control of restriction activity in vivo is discussed.

Bacterial Proteins↗

PubMed2005

Continuous assays for DNA translocation using fluorescent triplex dissociation: application to type I restriction endonucleases.

Fluorescent assays and accompanying kinetic models are described for the analysis of DNA translocation independent of duplex unwinding. A triplex binding site (TBS) was introduced into DNA substrates at precise loci downstream of recognition sequences for type IA, IB and IC restriction endonucleases (EcoKI, EcoAI and EcoR124I, respectively). Each endonuclease was incubated (without ATP) with substrates on which a hexachlorofluoroscein-labelled triplex-forming oligonucleotide (HEX-TFO) was pre-bound. Following addition of ATP, 1-D enzyme motion resulted in collision with, and displacement of, the HEX-TFO, producing a >twofold increase in fluorescent intensity. Alternatively, a decrease in anisotropy following displacement of a rhodamine-labelled TFO was monitored. Using rapid mixing in a stopped-flow fluorimeter, continuous kinetic profiles were produced in which displacement is preceded by a lag-phase, directly proportional to the distance moved. For each enzyme, we obtained not only the translocation rate but also information on slow isomerisation step(s) at initiation. Furthermore, we demonstrated that enzymes deficient in DNA cleavage but with maximal ATPase activity showed initiation and translocation rates identical to wild-type, confirming that DNA strand breaks are not a pre-requisite of motion.

Base Sequence↗

PubMed2004

DNA communications by Type III restriction endonucleases--confirmation of 1D translocation over 3D looping.

DNA cleavage by Type III restriction enzymes is governed strictly by the relative arrangement of recognition sites on a DNA substrate--endonuclease activity is usually only triggered by sequences in head-to-head orientation. Tens to thousands of base pairs can separate these sites. Long distance communication over such distances could occur by either one-dimensional (1D) DNA translocation or 3D DNA looping. To distinguish between these alternatives, we analysed the activity of EcoPI and EcoP15I on DNA catenanes in which the recognition sites were either on the same or separate rings. While substrates with a pair of sites located on the same ring were cleaved efficiently, catenanes with sites on separate rings were not cleaved. These results exclude a simple 3D DNA-looping activity. To characterize the interactions further, EcoPI was incubated with plasmids carrying two recognition sites interspersed with two 21res sites for site-specific recombination by Tn21 resolvase; inhibition of recombination would indicate the formation of stable DNA loops. No inhibition was observed, even under conditions where EcoPI translocation could also occur.

Anthracenes↗

PubMed2004

Enzyme-mediated DNA looping.

Most reactions on DNA are carried out by multimeric protein complexes that interact with two or more sites in the DNA and thus loop out the DNA between the sites. The enzymes that catalyze these reactions usually have no activity until they interact with both sites. This review examines the mechanisms for the assembly of protein complexes spanning two DNA sites and the resultant triggering of enzyme activity. There are two main routes for bringing together distant DNA sites in an enzyme complex: either the proteins bind concurrently to both sites and capture the intervening DNA in a loop, or they translocate the DNA between one site and another into an expanding loop, by an energy-dependent translocation mechanism. Both capture and translocation mechanisms are discussed here, with reference to the various types of restriction endonuclease that interact with two recognition sites before cleaving DNA.

Binding Sites↗

PubMed2003

S-adenosyl methionine prevents promiscuous DNA cleavage by the EcoP1I type III restriction enzyme.

DNA cleavage by the type III restriction endonuclease EcoP1I was analysed on circular and catenane DNA in a variety of buffers with different salts. In the presence of the cofactor S-adenosyl methionine (AdoMet), and irrespective of buffer, only substrates with two EcoP1I sites in inverted repeat were susceptible to cleavage. Maximal activity was achieved at a Res2Mod2 to site ratio of approximately 1:1 yet resulted in cleavage at only one of the two sites. In contrast, the outcome of reactions in the absence of AdoMet was dependent upon the identity of the monovalent buffer components, in particular the identity of the cation. With Na+, cleavage was observed only on substrates with two sites in inverted repeat at elevated enzyme to site ratios (>15:1). However, with K+ every substrate tested was susceptible to cleavage above an enzyme to site ratio of approximately 3:1, including a DNA molecule with two directly repeated sites and even a DNA molecule with a single site. Above an enzyme to site ratio of 2:1, substrates with two sites in inverted repeat were cleaved at both cognate sites. The rates of cleavage suggested two separate events: a fast primary reaction for the first cleavage of a pair of inverted sites; and an order-of-magnitude slower secondary reaction for the second cleavage of the pair or for the first cleavage of all other site combinations. EcoP1I enzymes mutated in either the ATPase or nuclease motifs did not produce the secondary cleavage reactions. Thus, AdoMet appears to play a dual role in type III endonuclease reactions: Firstly, as an allosteric activator, promoting DNA association; and secondly, as a "specificity factor", ensuring that cleavage occurs only when two endonucleases bind two recognition sites in a designated orientation. However, given the right conditions, AdoMet is not strictly required for DNA cleavage by a type III enzyme.

Adenosine Triphosphatases↗

PubMed2003

Evidence for DNA translocation by the ISWI chromatin-remodeling enzyme.

The ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin-remodeling activities. Here, we studied the interaction of the ISWI protein with nucleosomal substrates. We found that the ability of nucleic acids to bind and stimulate the ATPase activity of ISWI depends on length. We also found that ISWI is able to displace triplex-forming oligonucleotides efficiently when they are introduced at sites close to a nucleosome but successively less efficiently 30 to 60 bp from its edge. The ability of ISWI to direct triplex displacement was specifically impeded by the introduction of 5- or 10-bp gaps in the 3'-5' strand between the triplex and the nucleosome. In combination, these observations suggest that ISWI is a 3'-5'-strand-specific, ATP-dependent DNA translocase that may be capable of forcing DNA over the surface of nucleosomes.

Adenosine Triphosphatases↗

PubMed2002

How to get from A to B: strategies for analysing protein motion on DNA.

Essentially all genetic events require proteins to move from one location in a DNA polymer to another location in the same chain. A protein will seldom bind to a specific site in the DNA by colliding directly with that site. Instead, the protein will almost always collide first with a random site anywhere in the DNA and then migrate to the specific site by a facilitated-diffusion process that is constrained to the zone of that DNA molecule. Thereafter, many proteins bound to their target sites translocate in a specified direction along the DNA by a energy-dependent vectorial mechanism. This review will discuss some of the strategies that have been developed to analyse the motion of proteins on DNA, with respect to both the random diffusion processes involved in target-site location by DNA-binding proteins and the vectorial processes involved in unidirectional translocation along DNA.

DNA↗

PubMed2002

Kinetic models of translocation, head-on collision, and DNA cleavage by type I restriction endonucleases.

Digestion of linear DNA by type I restriction endonucleases is generally activated following the head-on collision of two translocating enzymes. However, the resulting distributions of cleavage loci along the DNA vary with different enzymes; in some cases, cleavage is located in a discrete region midway between a pair of recognition sites while in other cases cleavage is broadly distributed and occurs at nearly every intervening locus. Statistical models for DNA translocation, collision, and cleavage are described that can account for these observations and that are generally applicable to other DNA-based motor proteins. If translocation is processive (stepping forward is significantly more likely than DNA dissociation), then the linear distribution of an ensemble of proteins can be described simply using a Poisson relationship. The pattern of cleavage sites resulting from collision between two processive type I enzymes over a distance d can then be described by a binomial distribution with a standard deviation 0.5 x d1/2. Alternatively, if translocation is nonprocessive (stepping forward or dissociating become equally likely events), the linear distribution is described by a continuum of populated states and is thus extended. Comparisons of model data to the kinetics of DNA translocation and cleavage discount the nonprocessive model. Instead, the observed differences between enzymes are due to asynchronous events that occur upon collision. Therefore, type I restriction enzymes can be described as having processive DNA translocation but, in some cases, nonprocessive DNA cleavage.

Base Sequence↗