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

Monoclonal antibodies specific for the cyclobutane pyrimidine dimer (CPD) a re widely used for detection and quantification of DNA photolesions. Howeve r, the mechanisms of antigen binding by anti-CPD antibodies are little unde rstood. Here we report NMR analyses of antigen recognition by TDM-P, which is a mouse monoclonal antibody specific for the cis-syn-cyclobutane thymine dimer (T[c,s]T), P-31 NMR and surface plasmon resonance data indicated tha t the epitope recognized by TDM-P comprises hexadeoxy-nucleotides centered on the CPD, Chemical shift perturbations observed for TDM-P Fab upon bindin g to d(T[c,s]T) and d(TAT[c,s]TAT) were examined in order to identify the b inding sites for these antigen analogs. It was revealed that d(T[c,s]T) bin ds to the central part of the antibody-combining site, while the CPD-flanki ng nucleotides bind to the positively charged area of the V-H domain via el ectrostatic interactions. By applying a novel NMR method utilizing a pair o f spin-labeled DNA analogs, the orientation of DNA with respect to the anti gen-binding site was determined: CPD-containing oligonucleotides bind to TD M-2 in a crooked form, draping the 3'-side of the nucleotides onto the H1 a nd H3 segments, with the 5'-side on the H2 and L3 segments. These data prov ide valuable information for antibody engineering of TDM-2.