Recognition of DNA by thymine dimers specific antibodies

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DNA damage induced by ultraviolet (UV) light is considered to be a principal cause of mutation, neoplastic cellualr transformation and cell death. The major classes of UV-induced photoproducts are cis-syn cyclobutane pyrimidine dimers (CPDs) , pyrimidine (6-4) pyrimidone photoproducts [ (6-4) photoproducts] and the Dewar isomers formed between adjacent pyrimidines in DNA.In order to detect and quantitate these DNA photoproducts, several monoclonal antibodies have been established. We have been studying a series of monoclonal antibodies that specifically recognize either CPDs or (6-4) photoproducts.In order to understand the molecular recognitions of the antibodies for the photoproducts, we have cloned and sequenced the variable region genes. Comparing these sequences revealed that all four anti- (6-4) photoproducts antibodies were highly similar to one another, although there were some differences in potential antigen-contact regions. To assess the influences of these sequence differen … More ces at the structural level, computer models were constructed for these antibodies. Most of the sequence differences occurred in potential antigen contact regions, suggesting specific positions that might account for the observed differences in binding affinities and selectivities. Single-chain Fv derivatives of these antibodies were therefore constructed and characterized to provide an experimental system in which structure-function relationships can be tested. These derivatives could be isolated from E.coli using metal affinity chromatographic steps and possessed the same binding specificities as the parent monoclonal antibodies. The binding constants for octadeoxynucleotide containing either CPDs or (6-4) photoproducts as to both scFvs and corresponding Fabs were compared by means of BIAcore biosensor. The dissociation rate constants (kdiss) of scFv for the antigen analog was found to be almost the same as that of Fab, while the association rate constant (kass) of 2scFv was two-fold higher than that of Fab, These results indicate that the antigen binding region of the scFv seems to function as that of the intact antibody. Furthermore, elevated concentrations of NaCl decreased kass values of both Fab and scFv drastically, suggesting that these antibodies bind to oligonucleotide containing photoproducts through electrostatic interactions. Less