ABSTRACT
The structure-specific recognition protein SSRP1 plays a role in transcription and replication in the chromatin context. Mediated by its C-terminal high mobility group (HMG) box domain, SSRP1 binds DNA non-sequence specifically but recognizes certain DNA structures. Using acetic acid urea polyacrylamide gel electrophoresis and mass spectrometry, we have examined the phosphorylation of maize SSRP1 by protein kinase CK2 alpha. The kinase phosphorylated several amino acid residues in the C-terminal part of the SSRP1 protein. Two phosphorylation sites were mapped in the very C-terminal region next to the HMG box domain, and about seven sites are localized within the acidic domain. Circular dichroism showed that the phosphorylation of the two C-terminal sites by CK2 alpha resulted in a structural change in the region of HMG box domain, because the negative peak of the CD spectrum at 222 nm was decreased by approximately 10%. In parallel, the phosphorylation induced the recognition of UV-damaged DNA, whereas the non-phosphorylated protein does not discriminate between UV-damaged DNA and control DNA. The affinity of CK2 alpha-phosphorylated SSRP1 for the DNA correlates with the degree of UV-induced DNA damage. Moreover, maize SSRP1 can restore the increased UV-sensitivity of a yeast strain lacking the NHP6A/B HMG domain proteins to levels of the control strain. Collectively, these findings indicate a role for SSRP1 in the UV response of eukaryotic cells.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , DNA/radiation effects , High Mobility Group Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Transcriptional Elongation Factors , Ultraviolet Rays , Amino Acid Substitution , Base Sequence , Casein Kinase II , Circular Dichroism , DNA Damage , DNA Primers , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , High Mobility Group Proteins/chemistry , High Mobility Group Proteins/genetics , Humans , Kinetics , Phosphorylation , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Conformation , Protein Serine-Threonine Kinases/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Zea mays/metabolismABSTRACT
The expression of genes involved in C(4) photosynthesis in maize is under tight tissue-specific and light-dependent control. There is strong evidence that this control is at least in part brought about by DOF transcription factors binding to the respective promoters. We analyzed the interaction of DOF1 and DOF2 proteins with a functional and a cryptic endogenous binding site derived from the maize phosphoenolpyruvate carboxylase promoter (-300 bp region) in the nucleosomal context. Various DNA fragments comprising this promoter region were reconstituted into mononucleosomes from purified components, resulting in different positions of the DOF binding sites on the nucleosome surface. Binding of recombinant transcription factors to the different types of nucleosomes was examined using electrophoretic mobility shift assays. Changing the translational position of the binding site on the nucleosome surface strongly affected the efficiency of the interaction with the DOF factors. Deletion of individual recognition motifs revealed a positive impact of DOF protein binding to the main binding site on interactions with the cryptic binding site. The addition of the chromosomal high-mobility group (HMG) protein HMGB5 to the binding reaction mixture facilitated nucleosome binding of the transcription factor independent from the position of the recognition sites. The relevance of the data for the activation of the promoter in vivo is discussed.
Subject(s)
DNA-Binding Proteins/metabolism , HMGB Proteins/chemistry , Nucleosomes/metabolism , Plant Proteins/chemistry , Transcription Factors/metabolism , Zea mays/chemistry , Binding Sites , DNA, Plant/chemistry , DNA-Binding Proteins/chemistry , Electrophoretic Mobility Shift Assay/methods , HMGB Proteins/metabolism , Nucleosomes/chemistry , Plant Proteins/metabolism , Protein Binding , Structure-Activity Relationship , Transcription Factors/chemistryABSTRACT
The high mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that can contribute to transcriptional control by interaction with certain transcription factors. Using the transcription factor Dof2 and five different maize HMGB proteins, we have examined the specificity of the HMGB-transcription factor interaction. The HMG-box DNA binding domain of HMGB1 is sufficient for the interaction with Dof2. Although all tested HMGB proteins can interact with Dof2, the various HMGB proteins stimulate the binding of Dof2 to its DNA target site with different efficiencies. The HMGB5 protein is clearly the most potent facilitator of Dof2 DNA binding. Maximal stimulation of the DNA binding by the HMGB proteins requires association of HMGB and Dof2 prior to DNA binding. HMGB5 and Dof2 form a ternary complex with the DNA, but within the protein-DNA complex the interaction of HMGB5 and Dof2 is different from that in solution, as in contrast to the proteins in solution, they cannot be cross-linked with glutaraldehyde when bound to DNA. Phosphorylation of HMGB1 by protein kinase CK2 abolishes the interaction with Dof2 and the stimulation of Dof2 DNA binding. These findings indicate that transcription factors may recruit certain members of the HMGB family as assistant factors.
