Assessment of nucleotide excision repair protein binding forces by atomic force microscopy and optical trapping

Contents

1 Introduction

2 Specific Aims
2.1 DNA-Protein Binding
2.2 DNA Strength during UvrABC Binding
2.3 Optical Tweezers

3 Background and Significance
3.1 Dimerization
3.2 Nucleotide Excision Repair Pathway
3.3 Atomic Force Spectroscopy
3.4 Optical Trapping

4 Preliminary Studies
4.1 Force Spectroscopy
4.2 Dynamic Force Spectroscopy
4.3 DNA Stretching
4.4 Groove Binding

5 Research Design and Methods
5.1 Sample Preparation
5.1.1 DNA Damage
5.1.2 DNA Damage Quantification
5.1.3 Binding DNA to AFM Tip
5.1.4 Binding DNA to Polystyrene Bead
5.1.5 Protein Sample Preparation
5.1.6 Binding Protein to Slide
5.2 AFM Setup
5.3 Optical Trapping Setup
5.4 Expected Sources of Error
5.5 Experimental Analysis
5.5.1 DNA-Protein Rupture Forces
5.5.2 Force Spectroscopy

A Alternative Methods
A.1 Scanning Probe
A.2 Mutations
A.3 Species of Protein

1 Introduction

DNA is under constant repair from the damage being done from sources such as UV radiation, mutagenic chemicals, and errors made by the cell’s DNA replication mechanisms. The ability for a cell to identify and repair the damaged DNA is crucial for the cell to be able to successfully function and replicate. On a systemic scale the repair is essential for maintaining long term genomic stability. When these pathways fail the usual response is for the cell to die but in some instances the damage is done in a region that causes the cell to become carcinogenic. The DNA repair enzymes are responsible for finding and correcting these mistakes.

There are many different types of damage that can be done to DNA ranging from dimerization to depurination. Each of these types of damage requires a slightly different repair mechanism. The specific type of damage that is being investigated in this proposal is pyridine dimerization which usually occurs as the result of exposure to UV radiation. The repair pathway being nucleotide excision repair which involves either the replacement or removal of a region surrounding the damaged DNA. Problems in this pathway are important in pathological conditions such as xeroderma pigmentosum which causes the skin to be over sensitive to sun exposure and a high incidence of cancer.

Also genetic engineering utilizes deletion and insertion of DNA bases into various different cells. Understand- ing the pathways utilized to identify the structural changes that signify damage could be utilized to construct more sensitive repair proteins. Understanding the mechanisms of repair proteins to replace the damaged DNA with the correct segment could be utilized to develop faster more efficient ways for modifying bacteria and cells in beneficial ways.

Finally understanding the mechanisms of DNA damage and repair are useful from an evolutionary standpoint. For cells and organisms to be capable of genetic adaptations to environmental forces and consequently long term survival the repair mechanisms need to work well enough to keep the genome stable, but make mistakes often enough to allow for enough diversity for survival. The balance struck between these two goals is highly variant between species. A deeper understanding of the recognition and repair of damaged DNA could provide insights into the driving factors behind the evolutionary process

2 Specific Aims

The goal of this research program is to investigate DNA repair proteins, to investigate binding force of the DNA repair protein to the defective DNA molecules using the tools developed for determining the DNA-protein complex binding strength specifically atomic force microscopy and optical trapping developed in [1, 2, 3, 4] and the magnetic tweezers methods developed in [5, 6, 7]. Studies have been done to determine the stochiometry and pathways of DNA repair [8, 9, 10]

Specifically the repair enzyme I wish to investigate is the UvrABC complex a highly conserved repair pathway for UV damage done to DNA. This complex is ideal because a significant amount of research has already been done using various tools to determine structurally how the molecule appears to bind to and recognize DNA [10, 11, 12, 13]. The goal of my research will be to determine and how strongly the complex binds to the DNA, the time-scale for binding, and how the strength of the DNA molecule is affected while the enzyme is acting. The research should provide new insights into the forces involved in recognition and replacement in the repair pathway. should confirm and strengthen results obtained from other methods of investigation while offering a significant amount of new information about the forces involved.

2.1 DNA-Protein Binding

The first set of experiments will explore the protein to DNA binding force by use of Single Molecule Force Spec- troscopy (SMFS). The UvrABC complex consists of three separate proteins: UvrA, UvrB, UvrC that interact together to perform the functions of excision and repair. With this method the entire complex will not be investi- gated, just the UvrA and the UvrB which are responsible for damage recognition. The UV exposed dsDNA strand will be attached to the tip of the AFM and the UvrA2B2 heterotetramer protein will be linked to the surface of the stage. The stage will be slowly moved and the deflection of the AFM tip, representing the force that is being exerted, will be recorded. A better understanding of the binding forces should further elucidate the method for recognition and regions and mechanisms involved. The data would also demonstrate the sensitivity the UvrB protein has to the amount of damage done. The factors that will be modulated in the experiment are the rate of stage movement, the dsDNA segment used (both sequence and length), and the amount of UV damage done, and the linking polymers used to bind the DNA and protein to the instrument surfaces. These should provide information about the behavior of the protein in a variety of situations and isolate the DNA-protein binding from the other factors such as linker elasticity and electrostatic interactions between DNA segments and the protein.

2.2 DNA Strength during UvrABC Binding

The second set of experiments will measure the tensile properties of the DNA while the complex is repairing damage. The dsDNA molecule will be attached to the stage and the AFM tip. The molecule will be stretched until taut by moving the stage away from the tip. As the complex is added the tension will be measured by recording the deflection of the tip corresponding to damage recognition, unwinding, and excision. The experiment will be to perform this process in the presence of low concentrations of UvrABC to record the changes in structural properties during nucleotide excision repair. Given that neither the UvrD nor the DNA polymerase I are present the repair process will not be able to complete and the DNA strand will be left in a structurally weak state. This can be further investigate by simply performing the stretching properties of the DNA molecule before and after the complex is added. Since it is known that the rate at which the repair occurs is ATP dependent[11] the concentration of ATP will be modulated to verify the observed events have a concentration dependence. Additionally the concentration of the UvrABC complex will be kept in very low concentrations (≈nM) in order to assure that only one complex is interacting at a given time, single molecule sensitivity.

2.3 Optical Tweezers

The third specific aim is to repeat the measurements of the first two experiments using a different tool in order to verify the numbers obtained. All of the experiments done on the AFM setup will also be conducted using an optical tweezers setup. This will allow for the results to be corroborated and the errors from setup-specific sources to be minimized. Optical trapping methods will be used to bind the DNA to a polystyrene bead that can be held in a fixed position using a laser. The deflections of this bead can be used to measure the forces being exerted on the bead. Furthermore magnetic tweezers offer an even higher force resolution if in the event the forces observed are too small to be seen by either AFM or OT. The setup for a magnetic tweezer is similar to optical tweezers, but the bead is a magnetic material and a magnetic field is used to hold the bead in place. The distance the bead moves away from resting is related to the force being exerted. Finally magnetic tweezers offer the ability to measure twisting and and exert torque on the DNA strand, which we do not initially plan on doing, but could provide an interesting extension to the research.

3 Background and Significance

The failure of the mechanisms of DNA repair play a crucial role in many diseases including cancers. If these mechanisms were better understood they could be utilized to develop stronger treatments. There are 3 primary types of damage that is done to the DNA: depurination, deamination, and dimerization. Each of these types of damage occurs in a different way and requires different repair mechanisms to fix. This proposal only deals with UV induced dimerization.

3.1 Dimerization

When UV light or some chemicals interact with DNA, they can interact with a pyrimidine base (cytosine and thymine) in a photocycloaddition reaction to form a cyclobutane-type pyrimidine dimer as shown in Figure 1. The formation of this dimer means that the adjacent thymine bases are no longer bound to the adenine bases on the complementary DNA molecule but to themselves. This disrupts the double helix nature of DNA as shown in Figure 2. After this occurs a protein complex such as a DNA polymerase trying to unzip the DNA molecule would not be able to get past this spot and the functions of the cell dependent on this sequence would be unable to proceed[14].

illustration not visible in this excerpt

Figure 1: Cyclobutane-type Pyrimidine Dimer Formation between adjacent Thymine bases

illustration not visible in this excerpt

Figure 2: Change in DNA structure due to dimerization

3.2 Nucleotide Excision Repair Pathway

The nucleotide excision repair pathway is shared by a wide variety of species ranging from bacteria to mammals. The process is performed by several complexes known collectively as UvrABC. The three proteins involved UvrA, UvrB, and UvrC act co-operatively to track, locate the damaged region, and perform the excision on the DNA molecule. The tracking is done using a UvrA2B2 heterotetramer driven by UvrB to locate the sites of a structural deformation in the DNA such as one caused by dimerization shown in Figure 2. The interaction the UvrB has with the lesion causes the UvrA-B complex to change shape and unwind a portion of the DNA adjacent to the lesion. The unwound DNA wraps around UvrB-DNA complex which through a currently debated mechanism recruits UvrC which cleaves damaged section at the 4th or 5th phosphodiester bond 3′ to the lesion and cleaving the 8th phosphodiester bond 5′ to the site. The UvrBC-DNA complex formed is stable until the UvrD (a DNA helicase) binds and displaces the damage containing section of the strand. The gap in the strand is then filled by DNA polymerase I. The final steps of repair are completed by DNA ligase [10, 15, 13].

3.3 Atomic Force Spectroscopy

The general setup for atomic force microscopy involves the use of a flexible cantilever with a tip and mirror attached at the end. The tip is made out of a material that is predictively interactive with the sample being probed and the mirror acts to amplify the tiny motion of the cantilever tip to an easily measurable quantity because small deviations in position and orientation of the mirror correspond to a small angle change of the beam path of the laser. This small change in angle is scaled into a change in beam position by the distance away the detector is from the sample. The force is determined by back calculating the position change on the detector to a angle change on the mirror to a small position change in the cantilever. This position change can be translated into a force because the cantilever can be modeled as a spring where the amount of deformation is linearly proportional to the applied force.

illustration not visible in this excerpt

Figure 3: The slightly modified AFM setup for use in performing force spectroscopy measurements on DNAprotein complexes

3.4 Optical Trapping

The setup for optical trapping uses a polystyrene bead held in place by optical trapping forces. The optical trapping forces act to push the bead toward the focus of the laser. The distance the bead is away from the focus can be converted into the force that must be acting on the bead by use of simple models.

illustration not visible in this excerpt

Figure 4: The Optical Trapping Setup Being Used for this Experiment

4 Preliminary Studies

The use of AFM and optical tweezers for force spectroscopy provide piconewton force, nanometer distance, and millisecond time resolution. The selection or use of either method will be based on the approximate force of the interaction and the likely range of forces to be probed although for most of the experiments both setups will be used to collaborate the results obtained. The two methods provide a slightly different range and accuracy of mea- surements. Both models can be approximated using a spring model. The spring constant for the cantilever arm used in a similar DNA-protein interaction was [Abbildung in dieser Leseprobe nicht enthalten].Thisstiffnessincombinationwithasub-nanometer sensitivity and the ability to see displacements of up to 1 mm results in the ability to see forces from[Abbildung in dieser Leseprobe nicht enthalten] [[3]]. The Optical Tweezers setup has a much lower effective spring constant of [Abbildung in dieser Leseprobe nicht enthalten].Thisallowsforamuch higher sensitivity up to the subpiconewton range, but in combination with the beam-shape of the laser limits the maximum force accurately measurable is [Abbildung in dieser Leseprobe nicht enthalten] . The limit placed on both setups for maximum force is the strength of the linking molecule to the tip or DNA strand. The unbinding force for this is[Abbildung in dieser Leseprobe nicht enthalten]so forces close to or above this number cannot be easily probed. A nice diagram illustrating the differences using a spring model is shown in Figure 5.

Studies using other proteins bound to DNA have shown an interaction force ranging between 40-200pN [[16]]. The interaction force represents a mean of the results so values above the 200pN need to be probed to obtain the result. With this information the AFM becomes the more useful tool since the forces being probed could easily exceed the 200pN maximum for the optical tweezer instrument.

The standard sorts of forces that would be expected in a DNA-protein binding are electrostatic forces, dipoledipole forces, and the hydrogen bonding forces. Each of these forces represent different portions of the DNA and protein interacting and have different mechanical characteristics and strengths associated with it. Additionally the type of force is capable of elucidating information on the DNA sequence specificity of the binding.

4.1 Force Spectroscopy

Force spectroscopy involves recording the force being exerted on the instrument against the displacement of the stage. The effective result is you can obtain a sort of elasticity factor on the complex being probed.

illustration not visible in this excerpt

Figure 5: Comparison of Spring Model for AFM and OT

Electrostatic Interactions Positively charged amphiphilic proteins tend to interact with the negatively charged phosphate groups on the DNA. Although this interaction is nonspecific there are several transcription factor.

4.2 Dynamic Force Spectroscopy

Dynamic force spectroscopy operates moving the stage at a velocity and recording the force. This is in contrast to force spectroscopy which records the force at specific displacements. DFS is a better suited method for investigating many of the properties of the interaction since it is better aligned with the thermodynamic properties of the system. WIth a slowly varying force rate the thermal fluctuations in the system have a greater chance of spontaneously overcome the energy barrier for the bond. The DFS method allows for measurement of the lifetime of the complex, the thermal off rate, and can be used to investigate energy barriers in the transitional states of the complex[2]

4.3 DNA Stretching

Another type of experiment that has been performed on many similar substrates is the stretching of the DNA molecule between the instrument and the stage. The force versus distance response of the stretching is recorded and is fairly well understood. The DNA begins to change shape from the regular dsDNA to S-DNA which is 1.7x as long as the original strand. The winding of the DNA also begins to change as this transition is made which can cause some proteins that are sensitive to larger structure to unbind[5].

4.4 Groove Binding

Some of the DNA-protein interactions involve the peptide binding in grooves formed in the double stranded helical structure of the DNA. The two classes of groove binding are minor and major. Minor groove binding involves the interaction of several small charged particles in the groove of the double helix. This binding does little to change the conformation of the DNA molecule and operates on a weaker bond. The major groove binding consists of strong electrostatic interactions between the peptide and the backbone of the DNA that cause a larger conformational change in the DNA][3]. The groove binding mechanisms are also sensitive to the stretching and winding of the DNA described in the last section.

[...]