Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry Spring 5-5-2011 Protein-Nucleic Acid Interactions in Nuclease and Polymerases abdur rob Abdur Rob Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Part of the Chemistry Commons Recommended Citation rob, abdur, "Protein-Nucleic Acid Interactions in Nuclease and Polymerases." Dissertation, Georgia State University, 2011. https://scholarworks.gsu.edu/chemistry_diss/54 This Dissertation is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. PROTEIN - NUCLEIC ACID INTERACTIONS IN NUCLEASE AND POLYMERASES by ABDUR ROB Under the Direction of Professor Zhen Huang ABSTRACT DNA polymerase binds to the double stranded DNA and extends the primer strand by adding deoxyribonucletide to the 3’-end. Several reactions in the polymerase active site have been reported by Kornberg in addition to the polymerization. We observed DNA polymerase I can act as a pyrophosphatase and hydrolyze deoxyribonucletide. In performing the pyrophosphatase activity, DNA polymerase I requires to interact with RNA. RNA in general, was found to activate the DNA polymerase I as pyrophosphatase. This hydrolysis causes depletion of dNTP and inhibits DNA polymeration synthesis in vitro . In this RNA-dependent catalysis, DNA polymerase I catalyzes only dNTP but not rNTP. We have also observed that many other DNA polymerases have this type of the RNA-dependent pyrophosphatase activity. Our experimental data suggest that the exonuclease active sites most likely play the critical role in this RNA- dependent dNTP hydrolysis, which might have a broader impact on biological systems. On the basis of the crystal structure of a ternary complex of RNase H ( Bacillus halodurans ), DNA, and RNA, we have introduced the selenium modification at the 6-position of guanine (G) by replacing the oxygen ( Se G). The Se G has been incorporated into DNA (6 nt. - 6 nucleotides) by solid phase synthesis. The crystal structure and biochemical studies with the modified Se G-DNA indicate that the Se DNA can base-pair with the RNA substrate and serve as a template for the RNA hydrolysis. In the crystal structure, it has been observed that the selenium introduction causes shifting (or unwinding) of the G-C base pair by 0.3 Å. Furthermore, the Se-modification can significately enhance the phosphate backbone cleavage (over 1000 fold) of the RNA substrate, although the modifications are remotely located on the DNA bases. This enhancement in the catalytic step is probably attributed to the unwinding of the local duplex, which shifts scissile phosphate bond towards the enzyme active site. Our structural, kinetic and thermodynamic investigations suggest a novel mechanism of RNase H catalysis, which was revealed by the atom-specific selenium modification. INDEX WORDS: DNA polymerase, Klenow fragment, Selenium-modified DNA, RNA, Phasing and crystallization, Template, Substrate, Scissile bond, Km, Kcat , Kapp , Electrophoresis, Mass spectrometry, Pyrophosphorylosis, X-ray diffraction, MIR, MAD, SAD, Anomalous scattering, RNase H, Base pair shifting. PROTEIN - NUCLEIC ACID INTERACTIONS IN NUCLEASES AND POLYMERASES by ABDUR ROB A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Arts and Science Georgia State University 2011 Copyright by Abdur Rob 2011 PROTEIN - NUCLEIC ACID INTERACTIONS IN NUCLEASES AND POLYMERASES by ABDUR ROB Committee Chair: Dr. Zhen Huang Committee: Dr. Stuart Anthony Allison Dr. Yujun George Zheng Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University May 2011 iv While most are dream about success, winners wake up and work hard to achieve it To my son, Tanjeem Rhyne v ACKNOWLEDGEMENTS A dissertation only lists one author’s name, but no one could receive a Ph.D., nor should want to receive it, without the help of many others. Acknowledging them here is not nearly enough, but it is a start. Earning a Ph.D. degree is a long journey, mixed with excitement and pain; nobody can overcome without sincere assistance from others. Prof. Zhen Huang has been my advisor over the past five years and it would not be enough to say that he always appears to me as friend, philosopher, and guide. I am very much proud of and appreciate him, especially because he allowed me to work in two exciting projects. At the beginning, when I had very little idea about how to study a problem scientifically, he gave me ideas for projects that could build on the previous work and be tailored to my interests. As I gained experience, he gave me more and more freedom to explore the questions myself. He is always interested in my research and progresses and has suggestions for improvements. In particular, he has taught me the importance of verifying the significance of a result, no matter how exciting the first observation seems. I will always be thankful for the environment he has created for me to learn and to become a scientist. I have also learned enormously from the other members of our group. Drs. Jia Sheng and Josef Salon helped me in synthesizing DNA and RNA oligonucleotides whenever I need them, which helped me to keep my research forward. Drs. Julianne Caton-Williams, Lina Lin and Sarah Spenser provided me much scientific information and encouragement in many ways. I am offering special thanks to Dr. Jianhua Gan for introducing X-ray crystallography, a new research area for me, which helped me to understand the science in details. I am also thankful for the vi friendship and discussions with the other members of the lab during my time here, especially the Ph.D. graduate students, including Bo Zhang, Manindar Kaur, Sibo Jiang, Wen Zhang, Lilian Kamau, Huiyan Sun, and Xifang Liu. My time here has allowed me to meet many more people, more than I have space to mention, without whom the time I spent would not have been nearly as rewarding. vii TABLE OF CONTENTS ACKNOWLEDGEMENTS ………………………………………………………………….. v LIST OF TABLES Chapter 2………………………………………………………………………………………. xi Chapter 3………………………………………………………………………………………. xi LIST OF FIGURES Chapter 2………………………………………………………………………………………. xii Chapter 3………………………………………………………………………………………. xiv LISTS OF SCHEMES Chapter 2……………………………………………………………………………………… xvi Chapter 3……………………………………………………………………………………… xvi Chapter 1: Introduction to Protein-Nucleic Acid Interactions in Nulceases and Polymerases…………………………………………...………………………………………. 1 Chapter 2: RNA-dependent Pyrophosphatase Activity of DNA polymerase I…..………….... 7 2.1 Abstract ……………………………………………………………………………... 7 2.2 Introduction…………………………………………………………………….......... 9 2.3 Materials and Methods……………………………………………………………… 13 2.3.1 Synthesis and purification of oligonucleotides………………………………….. 13 2.3.2 Extraction of total RNA…………………………………………………............ 14 2.3.3 Synthesis of γ-³²P-dATP……………………………………………................... 14 2.3.4 Degradation reaction……………………………………………………………. 15 viii 2.3.5 Alkali hydrolysis………………………………………………………………... 15 2.3.6 Pyrophosphatase reaction………………………………………………………... 15 2.3.7 Polynucleotide kinase reaction……………………………………………........... 16 2.3.8 DNA polymerization reaction in the presence of RNA………………………… 16 2.3.9 TLC analysis……………………………………………………………….......... 17 2.3.10 FPLC analysis…………………………………………………………………. 17 2.3.11 Gel shift assay………………………………………………………………….. 18 2.3.12 Bacterial growth study………………………………………………………… 19 2.3.13 Mass spectrometry analysis……………………………………………………………. 19 2.3.14 Kinetic analysis………………………………………………………………………... 19 2.4 Results and discussions……………………………………………………………… 21 2.4.1 Klenow fragment hydrolyzes of dCTP into dCMP and PPi……………………. 22 2.4.2 Klenow fragment hydrolyzes of dATP into dAMP and PPi in presence of RNA…………………………………………………………………… 25 2.4.3 Anion exchange chromatography analysis shows dAMP as product in RNA-dependent dATP hydrolysis by DNA polymerase I……………………………… 27 2.4.4 Mass spectrometry showed dAMP as a product in RNA dependent dATP hydrolysis by Klenow fragment……………………………………………….. 34 2.4.5 The second product of dATP hydrolysis was pyrophosphate (PPi) in RNA dependent catalysis by Klenow fragment……………………………………. 36 2.4.6 The substrate of Klenow fragment in RNA dependent hydrolysis was only deoxyribonucleotides (dNTP’s)………………………………....................... 39 2.4.7 Binding of RNA with DNA polymerase I is fairly strong……………………… 48 2.4.8 The activator RNA follows Michaelis-Menten kinetics………………………... 52 ix 2.4.9 Hydrolysis of dNTP inhibits DNA polymerization ……………………………. 55 2.4.10 Pyrophosphate inhibits dNTP hydrolysis by DNA polymerase I in the presence of RNA………………………………………………… 58 2.4.11 All DNA polymerases interact with RNA and hydrolyze dNT ………………. 67 2.4.12 Three active sites of DNA polymerases involved in dNTP catalysis in the presence of RNA……………………………………………………... 70 2.4.13 DNA polymerases with three functional domains have higher catalytic activity in RNA dependent dNTP hydrolysis……………………………….. 71 2.4.14 RNA dependent dNTP hydrolysis by DNA polymerase caused bacterial growth inhibition …………………………………………………………… 89 2.5 Summary……………………………………………………………………………... 92 2.6 Conclusions………………………………………………………………………….. 96 Chapter 3: Kinetic Analysis of RNase H (BH) Enzyme with Native and