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

  Mader, Kevin S  

  Principal Investigator/Program Director(Last, First, Middle):  

  Contents  

1  Introduction  3

2  Specific Aims  3

2.1  DNA-Protein Binding  3

2.2  DNA Strength during UvrABC Binding  4

2.3  Optical Tweezers  4

3  Background and Significance  4

3.1  Dimerization  5

3.2  Nucleotide Excision Repair Pathway  6

3.3  Atomic Force Spectroscopy  6

3.4  Optical Trapping  7

4  Preliminary Studies  7

4.1  Force Spectroscopy  7

4.2  Dynamic Force Spectroscopy  8

4.3  DNA Stretching  8

4.4  Groove Binding  8

5  Research Design and Methods  9

5.1  Sample Preparation  9

5.1.1  DNA Damage  10

5.1.2  DNA Damage Quantification  10

5.1.3  Binding DNA to AFM Tip  10

5.1.4  Binding DNA to Polystyrene Bead  10

5.1.5  Protein Sample Preparation  10

5.1.6  Binding Protein to Slide  11

5.2  AFM Setup  11

5.3  Optical Trapping Setup  12

5.4  Expected Sources of Error  12

5.5  Experimental Analysis  13

5.5.1  DNA-Protein Rupture Forces  13

5.5.2  Force Spectroscopy  13

  A Alternative Methods  16

  A 1 Scanning Probe  16

  A 2 Mutations  16

  A 3 Species of Protein  16

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Principal Investigator/Program Director(Last, First, Middle):

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. Understanding 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 Spectroscopy (SMFS). The UvrABC complex consists of three separate proteins: UvrA, UvrB, UvrC that interact

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together to perform the functions of excision and repair. With this method the entire complex will not be investigated, 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 UvrA 2 B 2 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

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Principal Investigator/Program Director(Last, First, Middle):

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].

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

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