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Investigation of UVC induced DNA damage formation and photolyase catalyzed repair of cyclobutane pyrimidine dimers [Elektronische Ressource] / Lal Mohan Kundu

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Dissertation for the Doctor’s degree submitted to the Faculty of Chemistry and Pharmacy at the Ludwig-Maximilians-University Munich Investigation of UVC Induced DNA Damage Formation and Photolyase Catalyzed Repair of Cyclobutane Pyrimidine Dimers Lal Mohan Kundu Hooghly, West Bengal, India 2005 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 von Herrn Prof. Dr. Thomas Carell betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, am 01.02. 2005 Lal Mohan Kundu Dissertation eingereicht am 01.02. 2005 Gutachter: Prof. Dr. Thomas Carell Prof. Dr. Hendrik Zipse Mündliche Prüfung am 18.02. 2005 The present work was performed from September 2000 to February 2004, under the supervision of Prof. Dr. Thomas Carell in the Department of Chemistry, Philipps-University Marburg and Department of Chemistry and Pharmacy, Ludwig-Maximilians University Munich.

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Published 01 January 2005
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Dissertation for the Doctor’s degree
submitted to the Faculty of Chemistry and Pharmacy
at the Ludwig-Maximilians-University Munich



Investigation of UVC Induced DNA Damage Formation
and Photolyase Catalyzed Repair of Cyclobutane
Pyrimidine Dimers





Lal Mohan Kundu
Hooghly, West Bengal, India

2005


Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der
Promotionsordnung vom 29. Januar 1998 von Herrn Prof. Dr. Thomas Carell
betreut.



Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.

München, am 01.02. 2005


Lal Mohan Kundu



Dissertation eingereicht am 01.02. 2005

Gutachter: Prof. Dr. Thomas Carell Prof. Dr. Hendrik Zipse
Mündliche Prüfung am 18.02. 2005

The present work was performed from September 2000 to February 2004, under
the supervision of Prof. Dr. Thomas Carell in the Department of Chemistry,
Philipps-University Marburg and Department of Chemistry and Pharmacy,
Ludwig-Maximilians University Munich.










Dedicated to
my dear parents, my beloved brother, affectionate uncles and adorable
grandparents







ʺOur duty is to encourage every one in his struggle to live up to his own highest
idea, and strive at the same time to make the ideal as near as possible to the
Truth ʺ ------------Swami Vivekananda






Asato Ma Sadgamaya
Tamaso Ma Jyotir-Gamaya




‘’Science seldom proceeds in the straightforward logical manner imagined by
outsiders’’ ---------------James D. Watson






Acknowledgement

First of all, my heartiest thanks goes to Prof. Dr. Thomas Carell for offering me an
interesting and important research topic; for his sincere supervision, guidance
and valuable suggestions throughout this work; for his time and care in
correcting the thesis and also for the personal care and affection, which I received
from him and his wife Birgit.

I am very thankful to Prof. Dr. Hendrik Zipse for taking responsibility as the co-
referee and also for his time to correct the thesis.

I thank Prof. Dr. Ingo–Peter Lorenz, Prof. Dr. Heinz Langhals, Prof. Dr. Heinrich
Leonhardt and Prof. Dr. Thomas Lindel for their acceptance to take charge as
examiners.

I’m very thankful to Dr. Uwe Linne, for his constant cooperation and effective
collaboration. I also thank Prof. Dr. Alfred Batschauer for his generous help and
valuable collaboration through-out this work.

My sincere thanks to the mass spectrometry and NMR section of Phillips-
University Marburg and Ludwig-Maximillians-University Munich for providing me
the necessary help and instrumental facilities.

I’m grateful to all of my colleagues in the Carell group, both in Marburg and in
Munich, for their cooperation and helping hands in all directions. My heartiest
thanks to Ms. Alexandra Mees, for her warm friendship and extensive help in
correcting the thesis. I thank Ms. Simone Arnold, Mr. Sascha Breeger and Ms.
Friederike Grolle for their affection and friendship. My special thanks to Dr. David
Hammond and Mr. Ulrich Hennecke for their time and care in correcting the thesis.
Many thanks to Mr. Johannes Gierlich and Mr. Martin von Meltzer for their
essential help in solving computer related problems. I’m also thankful to Ms. Ina
Pinnschmidt, Ms. Katja Kraeling, Mrs. Slava Gaertner and Mrs. Sabine Voss for their
help and cooperation.

My sincere thanks to my dearest friends Judith, Srinivasa and Roopa, for their
unforgettable help, company and suggestions. My heartiest thanks to dear
Sukanya. Thanks to Kiran, Sabyashachi, Shyama and Subhashisda for their
friendship and help.

Above all, I owe to my parents Sabita and Krishna Chandra Kundu, brother Pintu,
uncle Bishnu Pada Mondal and Harisankar Kundu and all of my well wishers for
their love and blessings.



Kundu, L M
Table of Contents

SUMMARY...............................................................................................................1
ZUSAMMENFASSUNG ...............................................................................................4
1. INTRODUCTION.................................................................................................8
1.1. Consequence of ozone layer destruction................................................8
1.2. Effect of UV exposure...........................................................................10
1.3. Photoproducts ......................................................................................11
1.3.1. DNA, mutagenesis and cancer......................................................16
1.3.2. Photolesions and mutagenesis19
1.3.3. Structure-activity relationships of the photoproducts .....................23
1.4. Repair of DNA photoproducts...............................................................26
1.5. DNA photolyases..................................................................................30
1.5.1. The mechanism of action of CPD photolyase................................31
1.5.2. Electron transfer mechanism between CPD and photolyase.........35
1.5.3. Energy transfer between the cofactors..........................................37
1.5.4. The photoactivation of FAD ...........................................................40
1.5.5. Photolyase-substrate co-crystal study...........................................42
1.5.6. (6-4) photolyases...........................................................................43
1.6. Molecular Beacons ...............................................................................45
1.6.1. Principles of MBs46
1.6.2. Designing molecular beacons: ......................................................49
1.6.3. Kinetics and thermodynamics of MBs............................................51
1.6.4. Surface immobilized MBs..............................................................52
1.6.5. Applications of MBs.......................................................................54
1.7. Mass spectrometry for biomolecular analysis .......................................56
2. AIMS OF RESEARCH........................................................................................60
3. UV INDUCED DAMAGE FORMATION EVENT ........................................................62
3.1. HPLC methodology ..............................................................................65
3.2. Lesion formation on the different of nucleobases .................................66
3.3. Stability of photoproducts under various pH conditions .......................72
3.4. Enzymatic digestion and lesion analysis...............................................74
3.5. HPLC-ESI-MS for damage detection....................................................75
Kundu, L M
3.6. Characterization of photoproducts by HPLC-ESI-MS/MS.....................79
3.7. Dose-dependence study.......................................................................86
3.8. Sequence context.................................................................................87
3.9. Conformation-dependence on photoproduct formation ........................98
3.9.1. CD spectral analysis....................................................................100
3.10. Summary and conclusion ...................................................................107
4. PHOTOLYASE CATALYZED CPD-LESION REPAIR STUDY...................................110
4.1. Activities of CPD-photolyase ..............................................................110
4.2. Molecular beacon based photolyase-substrate DNA..........................111
4.2.1. Synthesis of the molecular beacon..............................................115
4.2.2. Characterization of the molecular beacon ...................................117
4.3. Activity profiling of Type-II photolyase with molecular beacon
substrate.............................................................................................121
4.3.1. HPLC profiling of photolyase activity ...........................................123
4.4. Repair study with Class-II photolyase from A. thaliana.......................126
4.5. Activity profiling of Class-II photolyase in crude cell extracts..............128
4.6. Study with living cell: single cell detection ..........................................131
4.7. Stability of the MB-probe in cell extract...............................................133
4.8. Summary and conclusion ...................................................................136
5. EXPERIMENTAL SECTION ..............................................................................138
5.1. Materials and Methods .......................................................................138
5.1.1. Equipments used in this study.....................................................138
5.1.2. Materials and Reagents...............................................................143
5.2. Methods..............................................................................................145
5.2.1. DNA damage study .....................................................................145
5.2.2. DNA repair...................................................................................154
6. ABBREVIATIONS...........................................................................................160
7. LITERATURE ................................................................................................162
Kundu, L M Summary
Summary

Gradual depletion of the ozone layer and consequently, increased ultraviolet
(UV) radiation on the Earth’s surface induces DNA-lesions inside the genome,
thereby causing mutations. Three kinds of photoproducts are mainly formed,
namely: cyclobutane pyrimidine dimers (CPD), pyrimidine-(6-4)-pyrimidone
dimer [(6-4)PP] and the Dewar valence isomer of (6-4)PP lesion. The formation of
these photolesions is a major cause of cell death (aging) and fatal disease like
skin cancer.

A part of this research was performed to investigate the formation and
characterization of DNA-lesions under UVC radiation. Small, fluorescent labeled
oligonucleotide hairpins (DNA, RNA and mixed DNA-RNA) were employed to
this purpose. The amount of damage was quantified using HPLC. Here, a new
method was developed, using ion-exchange SAX-column which works at
pH = 13, to measure the lesion formation in a direct way. In order to know which
lesions are formed, the irradiated hairpins were enzymatically digested. The
lesions were separated by HPLC followed by their characterization using MS/MS
fragmentation analysis.

Investigation was performed to understand the impact of the neighboring
nucleobases on the photo-reactivity of a dTpdT-dinucleotide. For this, hairpins
were prepared in various sequential contexts. Analysis of these irradiated
hairpins revealed the surprising result that the reactivity is strongly reduced
when a dTpdT dinucleotide is locked between two neighboring 2’-
deoxyguanosines, strongly implying that genomic DNA will be
inhomogeneously damaged and hence mutated under UVC radiation.
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