Discrimination of lesion removal of N-methylpurine-DNA glycosylase revealed by a potent neutralizing monoclonal antibody

Sanjay Adhikari, Stephen Kennel, Gargi Roy, Partha S. Mitra, Sankar Mitra, Rabindra Roy

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)31-39
Number of pages9
JournalDNA Repair
Volume7
Issue number1
DOIs
StatePublished - Jan 1 2008

Fingerprint

Neutralizing Antibodies
Epitopes
Monoclonal Antibodies
Substrates
DNA Repair Enzymes
Hypoxanthine
Surface Plasmon Resonance
Hybridomas
DNA Repair
Lipid Peroxidation
DNA Damage
Tail
Spectrum Analysis
Surface plasmon resonance
Clone Cells
Immunoglobulin G
Assays
Repair
Antibodies
Spectroscopy

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Discrimination of lesion removal of N-methylpurine-DNA glycosylase revealed by a potent neutralizing monoclonal antibody. / Adhikari, Sanjay; Kennel, Stephen; Roy, Gargi; Mitra, Partha S.; Mitra, Sankar; Roy, Rabindra.

In: DNA Repair, Vol. 7, No. 1, 01.01.2008, p. 31-39.

Research output: Contribution to journalArticle

Adhikari, Sanjay ; Kennel, Stephen ; Roy, Gargi ; Mitra, Partha S. ; Mitra, Sankar ; Roy, Rabindra. / Discrimination of lesion removal of N-methylpurine-DNA glycosylase revealed by a potent neutralizing monoclonal antibody. In: DNA Repair. 2008 ; Vol. 7, No. 1. pp. 31-39.
@article{b6204dc96a7b472a823f97db3302e3f3,
title = "Discrimination of lesion removal of N-methylpurine-DNA glycosylase revealed by a potent neutralizing monoclonal antibody",
abstract = "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.",
author = "Sanjay Adhikari and Stephen Kennel and Gargi Roy and Mitra, {Partha S.} and Sankar Mitra and Rabindra Roy",
year = "2008",
month = "1",
day = "1",
doi = "10.1016/j.dnarep.2007.07.012",
language = "English (US)",
volume = "7",
pages = "31--39",
journal = "DNA Repair",
issn = "1568-7864",
publisher = "Elsevier",
number = "1",

}

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

AU - Roy, Gargi

AU - Mitra, Partha S.

AU - Mitra, Sankar

AU - Roy, Rabindra

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.

UR - http://www.scopus.com/inward/record.url?scp=36549048414&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=36549048414&partnerID=8YFLogxK

U2 - 10.1016/j.dnarep.2007.07.012

DO - 10.1016/j.dnarep.2007.07.012

M3 - Article

VL - 7

SP - 31

EP - 39

JO - DNA Repair

JF - DNA Repair

SN - 1568-7864

IS - 1

ER -