Raquel Sánchez Giraldo
High Mobility Group proteins (HMG) are architectural factors involved in modulating chromatin structure and influencing a myriad of cellular processes such as transcription, replication and DNA repair. Altered levels of expression and mislocalization of these proteins have several pathological outcomes such as cancer and inflammatory processes. Understanding how the different HMG proteins recognize DNA and which changes can induce in it is still a challenge, and will be of great importance to interpret their activities in vivo. For this purpose, the aim of this work is to obtain the atomic structure of HMG proteins in complex with DNA using X-ray crystallography. In this work, we have studied two HMG families: HMGA and HMGB. Both bind to the minor groove of the DNA but are distinguished by their binding motifs: AT-hook motif (HMGA) and HMG-box motif (HMGB). AT-hooks are short positively charged flexible segments that bind preferentially to AT rich regions whereas HMG-box domains contain ~75 amino acids and have a characteristic L-shaped fold consisting of three a-helices and bind to DNA with limited or no sequence specificity. We have expressed and purified the HMGA1a protein, the fragment HMGA1b(1-79), the fragments Box A, Box B and Box A+B of the HMGB1 and the NHP6A (yeast HMGB). We have performed crystallographic assays of the complexes of AT rich oligonucleotides with these proteins (as well as with synthetic peptides corresponding to the AT-hook motif). We have studied the packing of some of the obtained crystals. As a major contribution of this thesis, we report the crystal structure of the HMGB1 Box A domain bound to the oligonucleotide ATATCGATAT at a resolution of 2 Å. It demonstrates a new mode of DNA recognition for HMG-box proteins. Two Box A domains act together kinking the DNA by 85º. In this unusual configuration the Phe37 of both domains stack together and intercalate the same CG base step. This duplex shows the greater distortion in just a base pair step that we have evidence for complexes with HMG-box domains.
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