title: Germ line variants of human N-methylpurine DNA glycosylase show impaired DNA repair activity and facilitate 1,N6-ethenoadenine-induced mutations.
abstract: Human N-methylpurine DNA glycosylase (hMPG) initiates base excision repair of a number of structurally diverse purine bases including 1,N(6)-ethenoadenine, hypoxanthine, and alkylation adducts in DNA. Genetic studies discovered at least eight validated non-synonymous single nucleotide polymorphisms (nsSNPs) of the hMPG gene in human populations that result in specific single amino acid substitutions. In this study, we tested the functional consequences of these nsSNPs of hMPG. Our results showed that two specific arginine residues, Arg-141 and Arg-120, are important for the activity of hMPG as the germ line variants R120C and R141Q had reduced enzymatic activity in vitro as well as in mammalian cells. Expression of these two variants in mammalian cells lacking endogenous MPG also showed an increase in mutations and sensitivity to an alkylating agent compared with the WT hMPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that these variants have substantial reduction in the equilibrium dissociation constant of binding (KD) of hMPG toward 1,N(6)-ethenoadenine-containing oligonucleotide (ϵA-DNA). Pre-steady-state kinetic studies showed that the substitutions at arginine residues affected the turnover of the enzyme significantly under multiple turnover condition. Surface plasmon resonance spectroscopy further showed that both variants had significantly decreased nonspecific (undamaged) DNA binding. Molecular modeling suggested that R141Q substitution may have resulted in a direct loss of the salt bridge between ϵA-DNA and hMPG, whereas R120C substitution redistributed, at a distance, the interactions among residues in the catalytic pocket. Together our results suggest that individuals carrying R120C and R141Q MPG variants may be at risk for genomic instability and associated diseases as a consequence.
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057,; Cancer Research Program, Houston Methodist Hospital Research Institute, Houston, Texas 77030, and.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Adhikari
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
authors.forename: Mahandranauth A
authors.initials: MA
authors.lastname: Chetram
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
authors.forename: Jordan
authors.initials: J
authors.lastname: Woodrick
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
authors.forename: Partha S
authors.initials: PS
authors.lastname: Mitra
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
authors.forename: Praveen V
authors.initials: PV
authors.lastname: Manthena
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
authors.forename: Pooja
authors.initials: P
authors.lastname: Khatkar
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
authors.forename: Sivanesan
authors.initials: S
authors.lastname: Dakshanamurthy
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
authors.forename: Monica
authors.initials: M
authors.lastname: Dixon
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
authors.forename: Soumendra K
authors.initials: SK
authors.lastname: Karmahapatra
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
authors.forename: Nikhil K
authors.initials: NK
authors.lastname: Nuthalapati
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
authors.forename: Suhani
authors.initials: S
authors.lastname: Gupta
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
authors.forename: Ganga
authors.initials: G
authors.lastname: Narasimhan
authors.affiliation: Department of Biochemistry and Molecular Medicine, The George Washington University, Washington, D. C. 20037.
authors.forename: Raja
authors.initials: R
authors.lastname: Mazumder
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Loffredo
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
authors.forename: Aykut
authors.initials: A
authors.lastname: Üren
authors.affiliation: From the Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057,. Electronic address: rr228@georgetown.edu.
authors.forename: Rabindra
authors.initials: R
authors.lastname: Roy
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D045647:DNA Glycosylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics; D015870:Gene Expression; D042822:Genomic Instability; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D009153:Mutagens / Q000494:pharmacology*; D020125:Mutation, Missense; D020641:Polymorphism, Single Nucleotide; D020349:Surface Plasmon Resonance
chemical_list: D009153:Mutagens; C004981:1,N(6)-ethenoadenine; D045647:DNA Glycosylases; D000225:Adenine
keywords: Base Excision Repair (BER); Carcinogenesis; DNA Damage; Genomic Instability; Single Nucleotide Polymorphism (SNP)