Nanofluidics for Static and Dynamic DNA-Protein Interaction Studies - Repair of Double-Strand Breaks from a Single-Molecule Perspective

Double-strand breaks (DSBs) is one of the most lethal forms of DNA damage. A single DSB may result in stalling of vital cellular machineries, and thus, requires immediate measures by the cell. Although the main hallmarks of DSB repair mechanisms are known for most prokaryotes and eukaryotes, details on crucial intermediate steps are still to be explained, such as how the free DNA ends are kept in close proximity during the repair process.\ua0\ua0\ua0\ua0\ua0\ua0\ua0\ua0 In the original work, upon which this Thesis is based, the two main DSB repair mechanisms, homologous recombination (HR) and non-homologous end-joining (NHEJ), have been studied from a molecular perspective. A fluorescence-based single-molecule nanofluidics assay has been developed and employed to characterize and visualize static biomolecular interactions between DNA and key DSB repairing proteins. Furthermore, a novel dynamic nanofluidic device has been developed to enable dynamic interaction studies in real time, allowing analytes to be introduced on-demand to stretched DNA molecules.\ua0\ua0\ua0\ua0\ua0\ua0\ua0\ua0 Single-molecule characterization of the NHEJ mechanism in Bacillus subtilis, comprising the Ku and Ligase D proteins, revealed that the end-joining activity is mediated by C-terminal protrusions on the homodimeric Ku complex. Using the novel dynamic nanofluidic device, the Ku homodimer was further demonstrated to stay bound to the DNA ends and junctions after completed repair, similar to the human Ku70/80 heterodimer. In addition, the traditional static nanofluidic device was used to identify a previously unknown potential DNA-bridging role of CtIP, a key protein in the human HR process. This could possibly explain how broken DNA ends are kept in close proximity during the initial steps of DSB repair through HR in humans. A similar method was employed to show that the Xrs2 component is indispensable for the end-joining activity of the Mre11-Rad50-Xrs2 complex of Saccharomyces cerevisiae

Single-Molecule Trapping and Measurement in a Nanostructured Lipid Bilayer System

The repulsive electrostatic force between a biomolecule and a like-charged surface can be geometrically tailored to create spatial traps for charged molecules in solution. Using a parallel-plate system composed of silicon dioxide surfaces, we recently demonstrated single-molecule trapping and high precision molecular charge measurements in a nanostructured free energy landscape. Here we show that surfaces coated with charged lipid bilayers provide a system with tunable surface properties for molecular electrometry experiments. Working with molecular species whose effective charge and geometry are well-defined, we demonstrate the ability to quantitatively probe the electrical charge density of a supported lipid bilayer. Our findings indicate that the fraction of charged lipids in nanoslit lipid bilayers can be significantly different from that in the precursor lipid mixtures used to generate them. We also explore the temporal stability of bilayer properties in nanofluidic systems. Beyond their relevance in molecular measurement, such experimental systems offer the opportunity to examine lipid bilayer formation and wetting dynamics on nanostructured surfaces

A nanofluidic device for real-time visualization of DNA–protein interactions on the single DNA molecule level

Single DNA molecule techniques have revolutionized our understanding of DNA-protein interactions. Traditional techniques for such studies have the major drawback that the DNA molecule studied is attached to a bead or a surface. Stretching of DNA molecules in nanofluidic channels has enabled single-molecule studies of DNA-protein interactions without the need of tethering the molecule to a foreign entity. This in turn allows for studying reactions along the whole extension of the molecule, including the free DNA ends. However, existing studies either rely on measurements where all components are mixed before introduction into the nanochannels or where passive diffusion brings the reagents to the confined DNA molecule. We here present a new generation of nanofluidic devices, where active exchange of the local environment within the nanofluidic channel is possible, while keeping the DNA molecule stretched and in confinement. To demonstrate the functionality of this novel device we added different analytes, such as SDS, spermidine and DNase I, to YOYO-1 stained DNA and studied the response in real time. We also performed a FRET-based reaction, where two different analytes were added sequentially to the same DNA molecule. We believe that this design will enable in situ mapping of complex biochemical processes, involving multiple proteins and cofactors, on single DNA molecules as well as other biomacromolecules

Phosphorylated CtIP bridges DNA to promote annealing of broken ends

The early steps of DNA double-strand break (DSB) repair in human cells involve the MRE11-RAD50-NBS1 (MRN) complex and its cofactor, phosphorylated CtIP. The roles of these proteins in nucleolytic DSB resection are well characterized, but their role in bridging the DNA ends for efficient and correct repair is much less explored. Here we study the binding of phosphorylated CtIP, which promotes the endonuclease activity of MRN, to single long (∼50 kb) DNA molecules using nanofluidic channels and compare it to the yeast homolog Sae2. CtIP bridges DNA in a manner that depends on the oligomeric state of the protein, and truncated mutants demonstrate that the bridging depends on CtIP regions distinct from those that stimulate the nuclease activity of MRN. Sae2 is a much smaller protein than CtIP, and its bridging is significantly less efficient. Our results demonstrate that the nuclease cofactor and structural functions of CtIP may depend on the same protein population, which may be crucial for CtIP functions in both homologous recombination and microhomology-mediated end-joining.ISSN:0027-8424ISSN:1091-649

Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging

Ionizing radiation (IR) is a common mode of cancer therapy, where DNA damage is the major reason of cell death. Here, we use an assay based on fluorescence imaging of single damaged DNA molecules isolated from radiated lymphocytes, to quantify IR induced DNA damage. The assay uses a cocktail of DNA-repair enzymes that recognizes and excises DNA lesions and then a polymerase and a ligase incorporate fluorescent nucleotides at the damage sites, resulting in a fluorescent “spot” at each site. The individual fluorescent spots can then be counted along single stretched DNA molecules and the global level of DNA damage can be quantified. Our results demonstrate that inclusion of the human apurinic/apyrimidinic endonuclease 1 (APE1) in the enzyme cocktail increases the sensitivity of the assay for detection of IR induced damage significantly. This optimized assay also allowed detection of a cooperative increase in DNA damage when IR was combined with mild hyperthermia, which is sometimes used as an adjuvant in IR therapy. Finally, we discuss how the method may be used to identify patients that are sensitive to IR and other types of DNA damaging agents

Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis

We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism

A Nanofluidic Device for Real-Time Visualization of DNA-Protein Interactions on the Single DNA Molecule Level

Single DNA molecule techniques have revolutionized our understanding of DNA-protein interactions. Traditional techniques for such studies have the major drawback that the DNA molecule studied is attached to a bead or a surface. Stretching of DNA molecules in nanofluidic channels has enabled single-molecule studies of DNA-protein interactions without the need of tethering the molecule to a foreign entity. This in turn allows for studying reactions along the whole extension of the molecule, including the free DNA ends. However, existing studies either rely on measurements where all components are mixed before introduction into the nanochannels or where passive diffusion brings the reagents to the confined DNA molecule. We here present a new generation of nanofluidic devices, where active exchange of the local environment within the nanofluidic channel is possible, while keeping the DNA molecule stretched and in confinement. To demonstrate the functionality of this novel device we added different analytes, such as SDS, spermidine and DNase I, to YOYO-1 stained DNA and studied the response in real time. We also performed a FRET-based reaction, where two different analytes were added sequentially to the same DNA molecule. We believe that this design will enable in situ mapping of complex biochemical processes, involving multiple proteins and cofactors, on single DNA molecules as well as other biomacromolecules