RESUMO
The high-mobility group protein HMGB1 contains two tandem DNA-binding HMG box domains, A and B, linked by a short flexible linker that allows the two domains to behave independently in the free protein. There is no structural information on how the linked domains and linker behave when bound to DNA, mainly due to the lack of any DNA-sequence preference of HMGB1. We report the structure determination, by NMR spectroscopy, of a well-defined complex of two tandem HMG boxes bound to a 16 bp oligonucleotide. The protein is an engineered version of the AB di-domain of HMGB1, in which the A box has been replaced by the HMG box of the sequence-specific transcription factor SRY, to give SRY.B. In the SRY.B/DNA complex, both HMG boxes bind in the minor groove and contribute to the overall DNA bending by intercalation of bulky hydrophobic residues between base-pairs; the bends reinforce each other, and the basic linker lies partly in the minor groove. As well as being the first structure of an HMG-box di-domain bound to DNA, this provides the first structure of the B domain of HMGB1 bound to DNA.
Assuntos
DNA/química , Domínios HMG-Box , Sequência de Aminoácidos , Escherichia coli/metabolismo , Proteína HMGB1/química , Proteínas de Grupo de Alta Mobilidade/química , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Oligonucleotídeos/química , Ligação Proteica , Homologia de Sequência de Aminoácidos , Proteína da Região Y Determinante do Sexo/químicaRESUMO
Lysine-63-linked polyubiquitin chains are not thought to signal protein degradation but instead signal for a variety of cellular processes including some types of DNA repair. RNA polymerase (Pol) II is polyubiquitinated following DNA damage or upon treatment of nuclear extracts with the transcription inhibitor alpha-amanitin. Here, we report, using a reaction in vitro, that transcription-dependent polyubiquitination of RNA Pol II consists of lysine-63-linked chains. This modification is specific for RNA Pol II engaged in active transcription and arrested by alpha-amanitin.
Assuntos
Lisina/metabolismo , Poliubiquitina/genética , Poliubiquitina/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Arginina/genética , Dano ao DNA , Ativação Enzimática/genética , Estabilidade Enzimática/genética , Humanos , Lisina/genéticaRESUMO
Transcription-coupled repair (TCR) is essential for the rapid, preferential removal of DNA damage in active genes. The large subunit of RNA polymerase (Pol) II is ubiquitinated in cells after UV-irradiation or cisplatin treatment, which induces DNA damage preferentially repaired by TCR. Several human mutations, such as Cockayne syndrome complementation groups A and B, are defective in TCR and incapable of Pol II ubiquitination upon DNA damage. Here we demonstrate a correlation between ubiquitination of RNA Pol II and arrest of transcription in vitro. Ubiquitination of Pol II is significantly induced by alpha-amanitin, an amatoxin that blocks Pol II elongation and causes its degradation in cells. Pol II undergoes similar ubiquitination on DNA containing cisplatin adducts that arrest transcription. Stimulation of ubiquitination requires the addition of template DNA and does not occur in the presence of an antibody to the general transcription factor TFIIB, indicating the transcription dependence of the reaction. We propose that components of the reaction recognize elongating Pol II-DNA complexes arrested by alpha-amanitin or cisplatin lesions, triggering ubiquitination.
