TY - JOUR
T1 - Discrimination of lesion removal of N-methylpurine-DNA glycosylase revealed by a potent neutralizing monoclonal antibody
AU - Adhikari, Sanjay
AU - Kennel, Stephen J.
AU - Roy, Gargi
AU - Mitra, Partha S.
AU - Mitra, Sankar
AU - Roy, Rabindra
N1 - Funding Information:
We thank Dr. Aykut Üren from Biacore Molecular interaction Shared Resources facility of the Lombardi Cancer Center for SPR studies. We thank Ms. Karen Howenstein for expert editorial and secretarial help. The work was supported by NIH grants RO1 CA 92306 (RR) and RO1 CA 53791 (SM).
Copyright:
Copyright 2009 Elsevier B.V., All rights reserved.
PY - 2008/1/1
Y1 - 2008/1/1
N2 - N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N-alkylpurine adducts, induced by alkylating chemotherapeutics, and deaminated and lipid peroxidation-induced purine adducts. We have generated monoclonal antibodies (moAbs) against human MPG. Twelve independent hybridoma clones were characterized, which, except 520-16A, are identical based on epitope exclusion assay. Four moAbs, including 520-2A, 520-3A, 520-16A, and 520-26A, have high affinity (KD ∼ 0.3-1.6 nM), and their subtypes were IgG2a, IgG1, IgG2a, and IgG2b, respectively. moAb 520-3A recognizes the sequence 52AQAPCPRERCLGPP66T, an epitope exclusively present in the N-terminal extension of human MPG. We found that moAb 520-3A significantly inhibited MPG's enzymatic activity towards different substrates, such as hypoxanthine, 1,N6ethenoadenine and methylated bases, which represent different classes of DNA damage, however, with different efficiencies. Real-time binding experiments using surface plasmon resonance (SPR) spectroscopy showed that the pronounced inhibition of activity was not in the substrate-binding step. Single turnover kinetics (STO) revealed that the inhibition was at the catalytic step. 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.
AB - N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N-alkylpurine adducts, induced by alkylating chemotherapeutics, and deaminated and lipid peroxidation-induced purine adducts. We have generated monoclonal antibodies (moAbs) against human MPG. Twelve independent hybridoma clones were characterized, which, except 520-16A, are identical based on epitope exclusion assay. Four moAbs, including 520-2A, 520-3A, 520-16A, and 520-26A, have high affinity (KD ∼ 0.3-1.6 nM), and their subtypes were IgG2a, IgG1, IgG2a, and IgG2b, respectively. moAb 520-3A recognizes the sequence 52AQAPCPRERCLGPP66T, an epitope exclusively present in the N-terminal extension of human MPG. We found that moAb 520-3A significantly inhibited MPG's enzymatic activity towards different substrates, such as hypoxanthine, 1,N6ethenoadenine and methylated bases, which represent different classes of DNA damage, however, with different efficiencies. Real-time binding experiments using surface plasmon resonance (SPR) spectroscopy showed that the pronounced inhibition of activity was not in the substrate-binding step. Single turnover kinetics (STO) revealed that the inhibition was at the catalytic step. 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.
KW - Base excision repair
KW - Epitope mapping
KW - Inhibition
KW - MPG
KW - Substrate specificity
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U2 - 10.1016/j.dnarep.2007.07.012
DO - 10.1016/j.dnarep.2007.07.012
M3 - Article
C2 - 17768096
AN - SCOPUS:36549048414
VL - 7
SP - 31
EP - 39
JO - DNA Repair
JF - DNA Repair
SN - 1568-7864
IS - 1
ER -