Effects of DNA sequence and structure on binding of RecA to single-stranded DNA

Fluorescence anisotropy is used to follow the binding of RecA to short sing le-stranded DNA (ssDNA) sequences (39 bases) at low DNA and RecA concentrat ion where the initial phase of polymerization occurs. We observe that RecA condensation is extremely sensitive to minute changes in DNA sequences. Rec A binds strongly to sequences that are rich in pyrimidines and that Pack si gnificant secondary structure and base stacking. We find a correlation betw een the DNA folding free energy and the onset concentration for RecA bindin g. These results suggest that the folding of ssDNA and base stacking repres ent a barrier for RecA binding. The link between secondary structure and bi nding affinity is further analyzed with two examples: discrimination betwee n two naturally occurring polymorphisms differing by one base and RecA bind ing on a molecular beacon. A self-assembly model is introduced to explain t hese observations. We propose that RecA may be used to sense ssDNA sequence and structure.