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Wheat HMGa protein is a typical member of the plant HMGA family. It has four AT hooks and a histone H1-like region. A panel of deletion mutants of HMGa was generated to study the role of different regions of HMGa in its binding to 4H (a synthetic DNA that mimics the in vivo structure of intermediates of homologous recombination and DNA repair) and linear DNAs. Although the histone H1-like region of HMGa does not bind to 4H or linear DNAs, it does enhance the binding. Mutants with any two adjacent AT hooks show specific binding to both 4H and linear P268 (and P31) with different binding affinities, which is partly due to the flanking regions between AT hooks. Conformational studies indicate that the alpha-helical content of HMGa increases significantly when it binds to 4H compared to that after binding to P31, linear DNA. In contrast, linear DNA, but not 4H, undergoes substantial conformational change when it binds to HMGa, indicating that linear DNA is relatively more flexible than 4H. A more significant difference in the affinities of binding of the mutants of HMGa to 4H was observed compared to their affinities of binding to linear DNA, P31. These differences could be due to the rigidity of the DNA and the characters of the AT hook regions in the mutants.