Regulations of DNA Alkylation/Deamination Damage Repair

Summary

Principal Investigator: Rabindra Roy
Abstract: Abstract Reactive nitrogen species, alkylating agents and lipid peroxide radicals generated endogenously and exogenously induce a myriad of DNA lesions, which is thought to affect genomic stability, cellular viability and cause multiple diseases such as cancer and aging. Such alkylated, deaminated and etheno adducts are generally repaired via an endogenous preventive pathway, base excision repair (BER), initiated when a DNA glycosylase removes the damaged base. Among these, a series of structurally diverse damaged purines are repaired by N- methylpurine DNA-glycosylase (MPG), present in all species from bacteria to man. Although a significant amount of information is available about the structure-function of mammalian MPG particularly due to efforts from our and other laboratories, the in vivo interactions of this enzyme which may profoundly affect its enzymatic activity, in vivo repair mode (patch size etc.), sequence specificity remains largely unknown. MPG physically interacts with and can be stimulated by various factors including hHR23A/B (a nucleotide excision repair protein) and XRCC1 (a BER protein). Moreover, our preliminary results show that BRCA1 directly interacts with and stimulates MPG's activity, whereas AP-endonuclease, the next enzyme in the same BER pathway binds several MPG substrate lesions without catalysis and inhibit MPG activity, and notably, not present in MPG pre-repair complex in the human cells. However, MPG lacking its N-terminal extension is stimulated by APE. Thus, these novel preliminary observations provide the ground work to test our central hypothesis that the dynamic protein-protein interactions or post-translational modification may modulate the MPG-mediated repair of spontaneous and induced alkylation, deamination and peroxidation-induced DNA damage to combat genomic instability and cancer. In our previous funding cycle we have developed a very precise and sensitive plasmid based in vivo method to monitor repair of [unreadable]A and Hx including intricate analysis of intermediate repair steps. In the next funding cycle, this repair assay method in combination with biochemical, proteomics and mammalian genetic approach (knock-out, mutant and siRNA knock-down) will be a valuable tool to identify genes involved in different steps of MPG-specific BER pathway and elucidate the repair mechanisms of [unreadable]A and Hx in vivo. Furthermore, direct protein-protein interactions in vitro and in vivo and detailed enzyme kinetics will also be used in order to understand a comprehensive mechanism of MPG-specific repair pathway(s) for [unreadable]A and Hx, which are representative of two different classes of DNA damaging agents. Our specific aims are to: (1) elucidate the molecular mechanisms of repair of [unreadable]`A and Hx inside the cells by determining the lesion-directed repair patch size, and repair efficiency depending on sequence context including mutation hotspot sequences in tumor suppressor gene, p53;(2) elucidate the mechanism of recognition of base lesions in MPG-specific BER pathway by analyzing the effect of BRCA1 in [unreadable]A and Hx repair in vivo and in vitro;and (3) elucidate the repair mechanisms subsequent to recognition and cleavage of base lesions in MPG-specific BER pathway by using various biochemical, proteomics, and mammalian genetic (knock-out, mutant and siRNA knock-down cells) approaches in combination with in vivo repair assay. Our long-term goal is comprehensive understanding of the role and regulation of MPG as a component of mammalian BER system for repair of alkylation, deamination, lipid-peroxidation- indiced DNA damage in human cells. The information from this study will also help to elucidate the function of other DNA glycosylases in BER pathway in combating various mutagenic and toxic DNA lesions in preventing cancer and aging. Furthermore, this knowledge will allow us eventually to devise strategies for modulating MPG expression for chemopreventive and therapeutic purposes.
Funding Period: 2001-04-01 - 2014-11-30
more information: NIH RePORT

Top Publications

  1. ncbi Magnesium, essential for base excision repair enzymes, inhibits substrate binding of N-methylpurine-DNA glycosylase
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
    J Biol Chem 281:29525-32. 2006
  2. pmc Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
    BMC Res Notes 5:134. 2012
  3. pmc A comparative study of recombinant mouse and human apurinic/apyrimidinic endonuclease
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
    Mol Cell Biochem 362:195-201. 2012
  4. pmc Development of a novel assay for human tyrosyl DNA phosphodiesterase 2
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
    Anal Biochem 416:112-6. 2011
  5. pmc Transcriptional regulation of the base excision repair pathway by BRCA1
    Tapas Saha
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA
    J Biol Chem 285:19092-105. 2010
  6. pmc A unified method for purification of basic proteins
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
    Anal Biochem 400:203-6. 2010
  7. pmc Excised damaged base determines the turnover of human N-methylpurine-DNA glycosylase
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States
    DNA Repair (Amst) 8:1201-6. 2009
  8. pmc Binding kinetics and activity of human poly(ADP-ribose) polymerase-1 on oligo-deoxyribonucleotide substrates
    Timothy J Jorgensen
    Departments of Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA
    J Mol Recognit 22:446-52. 2009
  9. ncbi Targeting base excision repair for chemosensitization
    Sanjay Adhikari
    Lombardi Comprehensive Cancer Center, 3800 Reservoir Road NW, Georgetown University Medical Center, Washington, DC 20057, USA
    Anticancer Agents Med Chem 8:351-7. 2008
  10. pmc Evidence of complete cellular repair of 1,N6-ethenoadenine, a mutagenic and potential damage for human cancer, revealed by a novel method
    Sujata Choudhury
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
    Mol Cell Biochem 313:19-28. 2008

Detail Information

Publications16

  1. ncbi Magnesium, essential for base excision repair enzymes, inhibits substrate binding of N-methylpurine-DNA glycosylase
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
    J Biol Chem 281:29525-32. 2006
    ....
  2. pmc Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
    BMC Res Notes 5:134. 2012
    ..Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg2+ in its activity was not studied in sufficient details...
  3. pmc A comparative study of recombinant mouse and human apurinic/apyrimidinic endonuclease
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
    Mol Cell Biochem 362:195-201. 2012
    ..This includes the development and validation of effective APE1 inhibitors as chemosensitizers in clinical studies...
  4. pmc Development of a novel assay for human tyrosyl DNA phosphodiesterase 2
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
    Anal Biochem 416:112-6. 2011
    ..Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner...
  5. pmc Transcriptional regulation of the base excision repair pathway by BRCA1
    Tapas Saha
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA
    J Biol Chem 285:19092-105. 2010
    ..These findings identify a novel mechanism through which BRCA1 may regulate the repair of oxidative DNA damage...
  6. pmc A unified method for purification of basic proteins
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
    Anal Biochem 400:203-6. 2010
    ..Approximately, two-fifths of human proteome, including many if not all nucleic acid-interacting proteins, have a pI of 7.94 or higher; virtually all these 12,000 proteins may be purified using this method in a single step...
  7. pmc Excised damaged base determines the turnover of human N-methylpurine-DNA glycosylase
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States
    DNA Repair (Amst) 8:1201-6. 2009
    ..Thus, the results provide the first evidence that the excised base rather than AP-site could be rate-limiting for DNA-glycosylase reactions...
  8. pmc Binding kinetics and activity of human poly(ADP-ribose) polymerase-1 on oligo-deoxyribonucleotide substrates
    Timothy J Jorgensen
    Departments of Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA
    J Mol Recognit 22:446-52. 2009
    ....
  9. ncbi Targeting base excision repair for chemosensitization
    Sanjay Adhikari
    Lombardi Comprehensive Cancer Center, 3800 Reservoir Road NW, Georgetown University Medical Center, Washington, DC 20057, USA
    Anticancer Agents Med Chem 8:351-7. 2008
    ..Thus, MPG and other BER proteins could be potential targets for chemosensitization...
  10. pmc Evidence of complete cellular repair of 1,N6-ethenoadenine, a mutagenic and potential damage for human cancer, revealed by a novel method
    Sujata Choudhury
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
    Mol Cell Biochem 313:19-28. 2008
    ..Furthermore, the epsilonA repair in vivo and in vitro is predominant in the G0/G1 phase of the cell cycle...
  11. pmc Expression, purification and characterization of codon-optimized human N-methylpurine-DNA glycosylase from Escherichia coli
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, LL level, S 122, 3800 Reservoir Road, NW, Washington, DC 20057, USA
    Protein Expr Purif 58:257-62. 2008
    ..Thus, this study indicates that this improved expression and purification system will facilitate large scale production and purification of a stable human MPG protein for further biochemical, biophysical and structure-function analysis...
  12. ncbi Dipole-dipole interaction stabilizes the transition state of apurinic/apyrimidinic endonuclease--abasic site interaction
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3800 Reservoir Road NW, Washington, D C 20057, USA
    J Biol Chem 283:1334-9. 2008
    ..Thus, low activation energy and the enthalpy of activation, which are perhaps a result of dipole-dipole interactions, play critical roles in AP site binding of APE...
  13. pmc Discrimination of lesion removal of N-methylpurine-DNA glycosylase revealed by a potent neutralizing monoclonal antibody
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States
    DNA Repair (Amst) 7:31-9. 2008
    ..Since we found that this antibody has an epitope in the N-terminal tail, the latter appears to have an important role in substrate discrimination, however, with a differential effect on different substrates...
  14. ncbi N-terminal extension of N-methylpurine DNA glycosylase is required for turnover in hypoxanthine excision reaction
    Sanjay Adhikari
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D C 20057, USA
    J Biol Chem 282:30078-84. 2007
    ..The results from this study also affirm the need for reinvestigation of full-length MPG for its enzymatic and structural properties, which are currently available mostly for the truncated protein...
  15. pmc Oxidative DNA damage repair in mammalian cells: a new perspective
    Tapas K Hazra
    Sealy Center for Molecular Science and Department of Biochemistry and Molecular Biology, 6 136 Medical Research Building, Route 1079, University of Texas Medical Branch, Galveston, TX 77555, USA
    DNA Repair (Amst) 6:470-80. 2007
    ....
  16. pmc Redox regulation of apurinic/apyrimidinic endonuclease 1 activity in Long-Evans Cinnamon rats during spontaneous hepatitis
    Soumendra Krishna Karmahapatra
    Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Georgetown University Medical Center, LL level, S 122 3800 Reservoir Road, NW, Washington, DC, 20057, USA
    Mol Cell Biochem 388:185-93. 2014
    ....

Research Grants30

  1. The Shelf Live Evaluation of Investigational Dosage Forms
    Jonathan White; Fiscal Year: 2013
    ..This contract is essential for continued assurance of the quality of drugs undergoing clinical investigation for different types of cancer by Cancer Therapeutics Evaluation Program. ..
  2. Spatial and Temporal Regulation of Angiogenesis
    HAROLD FISHER DVORAK; Fiscal Year: 2013
    ..abstract_text> ..
  3. The Virtual Physiological Rat Project
    Daniel A Beard; Fiscal Year: 2013
    ..This proposal targets the grand challenge of understanding complex multi-faceted disease phenotypes through experiments and simulations that capture the complex genotype-environment-phenotype relationship. ..
  4. Growth Factor Receptor in Breast Cancer Progression and Metabolic Regulation
    Mien Chie Hung; Fiscal Year: 2013
    ..abstract_text> ..
  5. Structural Cell Biology of DNA Repair Machines
    John A Tainer; Fiscal Year: 2013
    ..abstract_text> ..
  6. Structure and Function of DNA Repair Enzymes
    Susan S Wallace; Fiscal Year: 2013
    ..In addition to bioinformatics for all projects, Core A will also perform kinetics analysis for Projects 2-4. Core C will provide the administrative underpinnings for the project. ..
  7. Intra- and Extra-Chromosomal Probes for Mutagenesis by Carcinogens
    John M Essigmann; Fiscal Year: 2013
    ..Finally, we are adapting our mutation-analysis technology to probe mutational mechanisms of adducts replicated intrachromosomally in mammalian cells. ..
  8. University of Maryland Greenebaum Cancer Center Support Grant
    Kevin J Cullen; Fiscal Year: 2013
    ..Reflecting our remarkable and continued growth, UMGCC seeks to renew its CCSG to enhance and expand its efforts in high-quality and clinically relevant cancer research. ..
  9. Novel Targets in Cancer Chemotherapy: Chemical Biology of Guanine Alkylation
    Michael P Stone; Fiscal Year: 2013
    ....
  10. Genetic Analysis of the Breast Tumor Microenvironment
    Michael C Ostrowski; Fiscal Year: 2013
    ....
  11. Repair of Oxidatively Damaged Guanines
    A LIEN L LU-CHANG; Fiscal Year: 2013
    ..These studies will advance our understanding of carcinogenesis process and form the background work for the development of new anti-cancer drugs. ..
  12. Genotoxicity and Repair of Tobacco-Specific Nitrosamine DNA Adducts
    Thomas E Spratt; Fiscal Year: 2013
    ..The repair of O2-POB-dT via NER and BER will examined in vitro using synthetic oligodeoxynucleotides. The role of transcription-coupled NER for O6-POB-dG and O2- POB-dT will be probed with a novel modified host cell reactivation. ..
  13. Processing of Complex Lesions in the Mammalian Genome
    Randy J Legerski; Fiscal Year: 2013
    ..These approaches have excellent potential to yield useful technical and therapeutic advances in genetic manipulation. ..
  14. Roles of Lig3 and XRCC1 Genes in Genome Stability
    Alan E Tomkinson; Fiscal Year: 2013
    ..abstract_text> ..
  15. The MRAD9 Radioresistance Gene
    Howard B Lieberman; Fiscal Year: 2013
    ..abstract_text> ..
  16. Chemistry and Biology of Oxidized Purine Lesions in DNA
    SHEILA SUE DAVID; Fiscal Year: 2013
    ....
  17. Semi-volatile PCBs: Sources, Exposures, Toxicities
    Larry W Robertson; Fiscal Year: 2013
    ..These data and dietary studies in the last Aim will provide a scientific basis for risk assessment and advice for stakeholders with the ultimate goal to protect highly-exposed individuals and populations. ..
  18. Protein Dynamics in Enzymatic Catalysis
    Robert Callender; Fiscal Year: 2013
    ..The Equipment Core (Core A) supports the specialized comprehensive suite of instrumentation for the Program. The Administrative Core (Core B) administers the Program Project. ..
  19. Rocky Mountain Regional Center of Excellence or Biodefense and Emerging Infectiou
    John T Belisle; Fiscal Year: 2013
    ..abstract_text> ..
  20. Mechanisms of selective excision and oxidative repair of alkylated DNA
    Manel Camps; Fiscal Year: 2013
    ..Third, our structure-function studies on 3mA glycosylase repair are a necessary first step for the design of small molecule inhibitors as a way to enhance the cytotoxicity of methylating agents. ..