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265
Recombination repair of DNA damage in Escherichia coli and bacteriophage
, 1999
"... Damage reversal and one-strand repair..........................................................................................................753 Two-strand repair........................................................................................................................................ ..."
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Cited by 180 (3 self)
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Damage reversal and one-strand repair..........................................................................................................753 Two-strand repair...............................................................................................................................................753 Homologous recombination versus recombinational repair.........................................................................754 The two mechanisms of two-strand damage...................................................................................................755 The two recombinational repair pathways of E. coli.....................................................................................755 Frequency of two-strand lesions.......................................................................................................................756 Recombinational repair capacity of E. coli cells............................................................................................756 SOS Response: Reaction of E. coli to DNA Damage.........................................................................................756 Repair instead of DNA damage checkpoints: the prokaryotic strategy......................................................756 Organization of the SOS regulon.....................................................................................................................757 Levels of SOS induction.....................................................................................................................................758
Estimating the persistence length of a worm-like chain molecule from forceextension measurements
- Biophys.J
, 1999
"... ABSTRACT We describe a simple computation of the worm-like chain model and obtain the corresponding force-versusextension curve. We propose an improvement to the Marko and Siggia interpolation formula of Bustamante et al (Science 1994, 265:1599–1600) that is useful for fitting experimental data. We ..."
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Cited by 88 (0 self)
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ABSTRACT We describe a simple computation of the worm-like chain model and obtain the corresponding force-versusextension curve. We propose an improvement to the Marko and Siggia interpolation formula of Bustamante et al (Science 1994, 265:1599–1600) that is useful for fitting experimental data. We apply it to the experimental elasticity curve of single DNA molecules. Finally, we present a tool to study the agreement between the worm-like chain model and experiments.
Probing single-stranded dna conformation flexibility using fluorescence spectroscopy
- Biophysical Journal
, 2004
"... ABSTRACT Single-stranded DNA (ssDNA) is an essential intermediate in various DNA metabolic processes and interacts with a large number of proteins. Due to its flexibility, the conformations of ssDNA in solution can only be described using statistical approaches, such as flexibly jointed or worm-like ..."
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Cited by 59 (7 self)
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ABSTRACT Single-stranded DNA (ssDNA) is an essential intermediate in various DNA metabolic processes and interacts with a large number of proteins. Due to its flexibility, the conformations of ssDNA in solution can only be described using statistical approaches, such as flexibly jointed or worm-like chain models. However, there is limited data available to assess such models quantitatively, especially for describing the flexibility of short ssDNA and RNA. To address this issue, we performed FRET studies of a series of oligodeoxythymidylates, (dT)N, over a wide range of salt concentrations and chain lengths (10 # N # 70 nucleotides), which provide systematic constraints for testing theoretical models. Unlike in mechanical studies where available ssDNA conformations are averaged out during the time it takes to perform measurements, fluorescence lifetimes may act here as an internal clock that influences fluorescence signals depending on how fast the ssDNA conformations fluctuate. A reasonably good agreement could be obtained between our data and the worm-like chain model provided that limited relaxations of the ssDNA conformations occur within the fluorescence lifetime of the donor. The persistence length thus estimated ranges from 1.5 nm in 2 M NaCl to 3 nm in 25 mM NaCl.
Behavior of supercoiled DNA
- Biophys. J
, 1998
"... ABSTRACT We study DNA supercoiling in a quantitative fashion by micromanipulating single linear DNA molecules with a magnetic field gradient. By anchoring one end of the DNA to multiple sites on a magnetic bead and the other end to multiple sites on a glass surface, we were able to exert torsional c ..."
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Cited by 47 (5 self)
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ABSTRACT We study DNA supercoiling in a quantitative fashion by micromanipulating single linear DNA molecules with a magnetic field gradient. By anchoring one end of the DNA to multiple sites on a magnetic bead and the other end to multiple sites on a glass surface, we were able to exert torsional control on the DNA. A rotating magnetic field was used to induce rotation of the magnetic bead, and reversibly over- and underwind the molecule. The magnetic field was also used to increase or decrease the stretching force exerted by the magnetic bead on the DNA. The molecule’s degree of supercoiling could therefore be quantitatively controlled and monitored, and tethered-particle motion analysis allowed us to measure the stretching force acting on the DNA. Experimental results indicate that this is a very powerful technique for measuring forces at the picoscale. We studied the effect of stretching forces ranging from 0.01 pN to 100 pN on supercoiled DNA (�0.1 � � � 0.2) in a variety of ionic conditions. Other effects, such as stretching-relaxing hysteresis and the braiding of two DNA molecules, are discussed.
2001b. Sequence-specific detection of individual DNA strands using engineered nanopores
"... We describe biosensor elements that are capable of identifying individual DNA strands with single-base resolution. Each biosensor element consists of an individual DNA oligonucleotide covalently attached within the lumen of the α-hemolysin (αHL) pore to form a "DNA-nanopore". The binding ..."
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Cited by 46 (3 self)
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We describe biosensor elements that are capable of identifying individual DNA strands with single-base resolution. Each biosensor element consists of an individual DNA oligonucleotide covalently attached within the lumen of the α-hemolysin (αHL) pore to form a "DNA-nanopore". The binding of single-stranded DNA (ssDNA) molecules to the tethered DNA strand causes changes in the ionic current flowing through a nanopore. On the basis of DNA duplex lifetimes, the DNA-nanopores are able to discriminate between individual DNA strands up to 30 nucleotides in length differing by a single base substitution. This was exemplified by the detection of a drug resistance-conferring mutation in the reverse transcriptase gene of HIV. In addition, the approach was used to sequence a complete codon in an individual DNA strand tethered to a nanopore. The detection and characterization of individual RNA or DNA molecules is a thriving area of research 1-4 with potential diagnostic applications. For example, single polynucleotide molecules can be studied by electrical recordings in which RNA or DNA strands are driven by an applied potential through a single protein pore Here, we report the application of engineered "DNA-nanopores" to sense individual DNA strands with single-base resolution. Each nanopore was built by covalently attaching an individual ssDNA oligomer within the lumen of an engineered version of the αHL pore from Staphylococcus aureus Results and discussion Single-base mismatches influence the binding of individual DNA strands to a DNA-nanopore. Each DNA-nanopore carried an individual DNA oligonucleotide linked via a disulfide bridge to a cysteine residue at position 17 of one subunit of the heptameric pore The influence of the types and the positions of mismatches was further investigated by additional single-channel current recordings with H 6 (17C-oligo-1) 1 . We studied the interaction of oligo-1 with oligonucleotides carrying all possible mismatches in three different positions: 3′-TAAZ 2 TGG-5′, 3′-TAZ 3 GTGG-5′, and 3′-TAAGTGZ 4 -5′ with (Z n = A, C, G, T). The mean event lifetimes were obtained by lifetime histogram analysis and are summarized in
Laser-induced heating in optical traps
- Biophys. J
, 2003
"... ABSTRACT In an optical tweezers experiment intense laser light is tightly focused to intensities of MW/cm2 in order to apply forces to submicron particles or to measure mechanical properties of macromolecules. It is important to quantify potentially harmful or misleading heating effects due to the h ..."
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Cited by 37 (0 self)
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ABSTRACT In an optical tweezers experiment intense laser light is tightly focused to intensities of MW/cm2 in order to apply forces to submicron particles or to measure mechanical properties of macromolecules. It is important to quantify potentially harmful or misleading heating effects due to the high light intensities in biophysical experiments. We present a model that incorporates the geometry of the experiment in a physically correct manner, including heat generation by light absorption in the neighborhood of the focus, balanced by outward heat flow, and heat sinking by the glass surfaces of the sample chamber. This is in contrast to the earlier simple models assuming heat generation in the trapped particle only. We find that in the most common experimental circumstances, using micron-sized polystyrene or silica beads, absorption of the laser light in the solvent around the trapped particle, not in the particle itself, is the most important contribution to heating. To validate our model we measured the spectrum of the Brownian motion of trapped beads in water and in glycerol as a function of the trapping laser intensity. Heating both increases the thermal motion of the bead and decreases the viscosity of the medium. We measured that the temperature in the focus increased by 34.2 6 0.1 K/W with 1064-nm laser light for 2200-nm-diameter polystyrene beads in glycerol, 43.8 6 2.2 K/W for 840-nm polystyrene beads in glycerol, 41.1 6 0.7 K/W for 502-nm polystyrene beads in glycerol, and 7.7 6 1.2 K/W for 500-nm silica beads and 8.1 6 2.1 K/W for 444-nm silica beads in water. Furthermore, we observed that in glycerol the heating effect increased when the bead was trapped further away from the cover glass/glycerol interface as predicted by the model. We show that even though the heating effect in water is rather small it can have non-negligible effects on trap calibration in typical biophysical experimental circumstances and should be taken into consideration when laser powers of more than 100 mW are used.
Construction of multiple-beam optical traps with nanometer-resolution position sensing
- IEEE J Sel Top Quant
, 1996
"... Abstract — We describe the design and construction of two different types of multiple-beam optical tweezers, each equipped with nanometer-resolution position detectors. Multiple optical traps can be created either by splitting a laser beam in two parts, based on its polarization, or time-sharing a s ..."
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Cited by 36 (1 self)
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Abstract — We describe the design and construction of two different types of multiple-beam optical tweezers, each equipped with nanometer-resolution position detectors. Multiple optical traps can be created either by splitting a laser beam in two parts, based on its polarization, or time-sharing a single beam among several different locations. The advantages and disadvan-tages of optical tweezers based on either scheme are discussed, along with details of specific implementations. Various ways to detect microscopic movements of an optically trapped object are presented and compared, including designs that are relatively insensitive to absolute location of a trapped particle within the field of view. Two of many possible applications for such instruments are illustrated: the detection of molecular steps by kinesin motor molecules, and determinations of the stiffness of single microtubules. I.
Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins. Nucleic Acids Res 34: 593–605
, 2006
"... The Gag polyprotein of HIV-1 is essential for retro-viral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interac-tion of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic ..."
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Cited by 31 (8 self)
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The Gag polyprotein of HIV-1 is essential for retro-viral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interac-tion of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experi-ments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be 10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are con-sistent with the primary function of Gag as a nucleic
Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion
- Nucleic Acids Research
"... The tethered particle motion (TPM) allows the direct detection of activity of a variety of biomolecules at the single molecule level. First pioneered for RNA polymerase, it has recently been applied also to other enzymes. In this work we employ TPM for a systematic investigation of the kinetics of D ..."
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Cited by 27 (7 self)
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The tethered particle motion (TPM) allows the direct detection of activity of a variety of biomolecules at the single molecule level. First pioneered for RNA polymerase, it has recently been applied also to other enzymes. In this work we employ TPM for a systematic investigation of the kinetics of DNA looping by wild-type Lac repressor (wt-LacI) and by hinge mutants Q60G and Q60 1 1. We implement a novel method for TPM data analysis to reliably measure the kinetics of loop formation and disrup-tion and to quantify the effects of the protein hinge flexibility and of DNA loop strain on such kinetics. We demonstrate that the flexibility of the protein hinge has a profound effect on the lifetime of the looped state. Our measurements also show that the DNA bending energy plays a minor role on loop disruption kinetics, while a strong effect is seen on the kinetics of loop formation. These observations substantiate the growing number of theoretical studies aimed at characterizing the effects of DNA flexibility, tension and torsion on the kinetics of protein binding and dissociation, strengthening the idea that these mechanical factors in vivo may play an important role in the modulation of gene expression regulation.
Sizing DNA using a nanometer-diameter pore
- Biophys. J. 2004
"... ABSTRACT Each species from bacteria to human has a distinct genetic fingerprint. Therefore, a mechanism that detects a single molecule of DNA represents the ultimate analytical tool. As a first step in the development of such a tool, we have explored using a nanometer-diameter pore, sputtered in a n ..."
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Cited by 24 (4 self)
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ABSTRACT Each species from bacteria to human has a distinct genetic fingerprint. Therefore, a mechanism that detects a single molecule of DNA represents the ultimate analytical tool. As a first step in the development of such a tool, we have explored using a nanometer-diameter pore, sputtered in a nanometer-thick inorganic membrane with a tightly focused electron beam, as a transducer that detects single molecules of DNA and produces an electrical signature of the structure. When an electric field is applied across the membrane, a DNA molecule immersed in electrolyte is attracted to the pore, blocks the current through it, and eventually translocates across the membrane as verified unequivocally by gel electrophoresis. The relationship between DNA translocation and blocking current has been established through molecular dynamics simulations. By measuring the duration and magnitude of the blocking current transient, we can discriminate single-stranded from double-stranded DNA and resolve the length of the polymer.
