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 ?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 ?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 ?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 ?`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 ?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.

Public Health Relevance

Damage to cellular DNA causes mutations and development of cancer and neurodegenaration. The DNA in human cell undergoes several thousand to million damaging events per day, generated by both environmental pollutants including tobacco smoke and internal metabolic (endogenous) processes. Such DNA adducts are repaired by an endogenous preventive pathway, Base Excision Repair (BER). The goal of this project is to understand the mechanisms of regulation of BER pathway and devise strategies for modulating the expression of BER genes to improve the efficacy of chemopreventives and therapeutics.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Cancer Etiology Study Section (CE)
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Okano, Paul
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Georgetown University
Internal Medicine/Medicine
Schools of Medicine
United States
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Woodrick, Jordan; Gupta, Suhani; Khatkar, Pooja et al. (2014) Slow repair of lipid peroxidation-induced DNA damage at p53 mutation hotspots in human cells caused by low turnover of a DNA glycosylase. Nucleic Acids Res 42:9033-46
Karmahapatra, Soumendra Krishna; Saha, Tapas; Adhikari, Sanjay et al. (2014) Redox regulation of apurinic/apyrimidinic endonuclease 1 activity in Long-Evans Cinnamon rats during spontaneous hepatitis. Mol Cell Biochem 388:185-93
Adhikari, Sanjay; Karmahapatra, Soumendra K; Karve, Tejaswita M et al. (2012) Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction. BMC Res Notes 5:134
Adhikari, Sanjay; Manthena, Praveen Varma; Kota, Krishna Kiran et al. (2012) A comparative study of recombinant mouse and human apurinic/apyrimidinic endonuclease. Mol Cell Biochem 362:195-201
Adhikari, Sanjay; Karmahapatra, Soumendra K; Elias, Hadi et al. (2011) Development of a novel assay for human tyrosyl DNA phosphodiesterase 2. Anal Biochem 416:112-6
Adhikari, Sanjay; Manthena, Praveen Varma; Sajwan, Kamal et al. (2010) A unified method for purification of basic proteins. Anal Biochem 400:203-6
Adhikari, Sanjay; Uren, Aykut; Roy, Rabindra (2009) Excised damaged base determines the turnover of human N-methylpurine-DNA glycosylase. DNA Repair (Amst) 8:1201-6
Adhikari, Sanjay; Manthena, Praveen Varma; Uren, Aykut et al. (2008) Expression, purification and characterization of codon-optimized human N-methylpurine-DNA glycosylase from Escherichia coli. Protein Expr Purif 58:257-62
Adhikari, Sanjay; Kennel, Stephen J; Roy, Gargi et al. (2008) Discrimination of lesion removal of N-methylpurine-DNA glycosylase revealed by a potent neutralizing monoclonal antibody. DNA Repair (Amst) 7:31-9
Adhikari, Sanjay; Uren, Aykut; Roy, Rabindra (2008) Dipole-dipole interaction stabilizes the transition state of apurinic/apyrimidinic endonuclease--abasic site interaction. J Biol Chem 283:1334-9

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