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Single-molecule investigations of nucleosomal DNA molecules as drug targets

Single-molecule investigations of nucleosomal DNA molecules as drug targets

  • 문현민 (대학원 물리학과 비선형동력학 및 생물물리학전공)

초록/요약

Cancer is one of the most common public health threats of the 21st century. It is a clinically diverse set of diseases affecting multicellular organisms that can appear in various phenotypes. Such diseases are caused by the accumulation of numerous genetic alterations in the cell, resulting in changes in gene expression patterns. There are three main routes of treatment to treat cancer: surgery, radiation therapy, and chemotherapy. Chemotherapy is used to reduce the size of cancer cells prior to surgery and radiation therapy. It is also used to prevent recurrence of cancer cells after surgery and radiation therapy. Chemotherapy is also used in palliative care for patients with terminal cancer to prolong survival and relieve symptoms. Above all, unlike surgery and radiotherapy, chemotherapy has the advantage of affecting the whole body through systemic delivery. Therefore, it is used for metastatic treatment in patients who are thought to have a potential for secondary growth but have not yet progressed clinically. Among chemotherapy drugs widely used for cancers, cisplatin is one of the most compelling drugs. Cisplatin, in its neutral state, passively diffuses across a cellular lipid bilayer, which is impermeable to charged particles. Since the intracellular concentration of Cl− is less than one tenth of its extracellular value, cisplatin becomes hydrolyzed into monovalent or divalent cations by releasing Cl− upon cell entry. The cationic state of cisplatin, which accumulates inside a cell, is the active form that binds to DNA. However, other anionic species such as carbonates, phosphates, and thiolates that are common inside cells are also known to interfere with cisplatin. Another important factor that deserves due attention is the complexity originating from packaging of DNA into chromatin, the form that cellular DNA adopts. Recent studies emphasized multi-faceted involvements of chromatin in various events triggered by cisplatin. Therefore, we have studied the effect of cisplatin on the nucleosomal DNA, as a chemotherapeutic subject, under physiological salt condition. To that end, we improved the magnetic tweezers and achieved nanometer resolution. Nucleosomal DNA was also prepared in vitro using Nucleosome Assembly Protein 1 (NAP1). Using this, we were able to directly observe changes in nucleosomal DNA due to physical forces or salts at high concentrations. We then treated the nucleosomal DNA with cisplatin in a physiological solution. When mechanical force or charge screening effects were applied to cisplatin treated nucleosomal DNA, we surprisingly observed suppression of core histone relaxation from DNA binding sites even a low concentration of cisplatin. Since chromatin is the basic setting for various genetic events and thus plays important roles in cellular metabolism, we tested another drug implicated to interfere with chromatin metabolism in the opposite way by destabilizing chromatin and nucleosome core particle. The drug is a curaxin, which has been known to simultaneously activate p53, inhibit NF-kB, and cause death of various tumor cells. It has also been shown that curaxin binds directly to DNA but does not damage DNA. According to a recent speculation about the effect of curaxin on chromatin and nucleosomal DNA, we tested it and found that the chromatin structure was destroyed and that the physical properties of curaxin-treated DNA was dramatically changed. This effect could disrupt various cellular processes and eventually cause cell death. The mechanistic details gained at single molecule level clarify the mechanism for the anti-cancer effect of chemotherapy drugs and confirm that the nucleosomal DNA is indeed effective as a drug target. This will be beneficial to designing a new generation of anti-cancer drugs.

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목차

1. INTRODUCTION 1
2. CHEMOTHERAPY DRUGS 8
2.1. CISPLATIN 10
2.1.1. Historical overview 10
2.1.2. Structure and properties 12
2.1.3. Molecular Biological mechanism (into the cell and forming DNA adduct) 13
2.2. CURAXIN CBL0137 15
3. NUCLEOSOMAL DNA CONSTRUCTED BY NUCLEOSOME ASSEMBLY PROTEIN1(NAP1) 17
3.1. STRUCTURE OF DNA 19
3.2. HISTONE 21
3.3. NUCLEOSOME ASSEMBLY PROTEIN1(NAP1) 22
4. MAGNETIC TWEEZERS 24
4.1. OVERVIEW OF MAGNETIC TWEEZERS 25
4.2. APPARATUS OF MAGNETIC TWEEZERS 27
4.3. SAMPLE PREPARATION 30
4.3.1. Preparation of a flow cell 30
4.3.1.1. Cleaning of the coverslip 30
4.3.1.2. Functionalization of the coverslip 31
4.3.1.3. Assembly of flow cell 32
4.3.2. Preparation of DNA samples for a magnetic tweezers experiment 32
4.3.2.1. Prepare plasmid DNA 33
4.3.2.2. Preparation of the Biotin- and Dibenzocyclooctyl (DBCO)- linkers 33
4.3.2.3. Enzyme cut each part of sample tether 36
4.3.2.4. Ligation of three fragments 38
4.3.3. Experimental chamber 39
4.3.3.1. Azide coating and passivation 39
4.3.3.2. Preparation of DNA sample with magnetic bead 41
4.4. DATA ACQUISITION AND ANALYSIS 42
4.4.1. Construction of the radial intensity profile from a diffraction ring pattern 42
4.4.2. Determination of the bead position in a 2-dimensional space 44
4.4.3. Determination of the z-axis position of the bead 47
4.5. CALIBRATION OF THE FORCE APPLIED TO A TETHERED MOLECULE 54
4.6. PULLING DNA : WLC MODEL 59
4.6.1. The KratkyPorod model 60
4.6.2. The worm like chain model 60
5. THE EFFECT OF CHEMOTHERAPY DRUGS ON NUCLEOSOMAL DNA 62
5.1. INTRODUCTION 62
5.2. METHODS 67
5.2.1. Invitro bulk assay (gel electrophoresis) 67
5.2.2. Nucleosome assembly assay 67
5.2.3. Magnetic Tweezser 68
5.3. RESULTS AND DISCUSSION 70
5.3.1. The degree of DNA platination by cisplatin under various salt conditions 70
5.3.2. Salt-sensitive Binding Affinity of Cisplatin to DNA Estimated from Direct Measurement of DNA Elasticity 74
5.3.3. Efficient Structural Fixation of the N-DNA by Cisplatin in Physiological Salt Conditions. 79
5.3.4. Curaxin disrupts nucleosomal DNA and changes the torsional property of DNA. 95
5.4. CONCLUSIONS 102
6. SUMMARY 110
7. REFERENCES 114
8. ACKNOWLEDGMENTS 124
9. 국문 초록 126
10. APPENDIX : MATLAB CODES 130
10.1. MTD_ANALYSIS.M : TO EXTRACT DATA FROM RAW FILES 130
10.2. DAT_LUT_ANALYSIS.M : TO ANALYZE THE EXTRACTED DATA 136

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