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Nucleic Acids Research, 2000, Vol. 28, No. 4 944-951
© 2000 Oxford University Press

DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer

Takuya Torizawa, Nobuhiro Yamamoto, Tomonori Suzuki1, Kaoru Nobuoka1, Yasuo Komatsu1, Hiroshi Morioka1, Osamu Nikaido2, Eiko Ohtsuka1, Koichi Kato and Ichio Shimada*

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan, 1Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan and 2Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-0934, Japan

Monoclonal antibodies specific for the cyclobutane pyrimidine dimer (CPD) are widely used for detection and quantification of DNA photolesions. However, the mechanisms of antigen binding by anti-CPD antibodies are little understood. Here we report NMR analyses of antigen recognition by TDM-2, which is a mouse monoclonal antibody specific for the cis-syn-cyclobutane thymine dimer (T[c,s]T). 31P NMR and surface plasmon resonance data indicated that the epitope recognized by TDM-2 comprises hexadeoxynucleotides centered on the CPD. Chemical shift perturbations observed for TDM-2 Fab upon binding to d(T[c,s]T) and d(TAT[c,s]TAT) were examined in order to identify the binding sites for these antigen analogs. It was revealed that d(T[c,s]T) binds to the central part of the antibody-combining site, while the CPD-flanking nucleotides bind to the positively charged area of the VH domain via electrostatic interactions. By applying a novel NMR method utilizing a pair of spin-labeled DNA analogs, the orientation of DNA with respect to the antigen-binding site was determined: CPD-containing oligonucleotides bind to TDM-2 in a crooked form, draping the 3'-side of the nucleotides onto the H1 and H3 segments, with the 5'-side on the H2 and L3 segments. These data provide valuable information for antibody engineering of TDM-2.

* To whom correspondence should be addressed. Tel: +81 3 5841 4810; Fax: +81 3 3815 6540; Email: shimada@iw-nmr.f.u-tokyo.ac.jp


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T. Torizawa, T. Ueda, S. Kuramitsu, K. Hitomi, T. Todo, S. Iwai, K. Morikawa, and I. Shimada
Investigation of the Cyclobutane Pyrimidine Dimer (CPD) Photolyase DNA Recognition Mechanism by NMR Analyses
J. Biol. Chem., July 30, 2004; 279(31): 32950 - 32956.
[Abstract] [Full Text] [PDF]


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