RESUMO
Deinococcus ficus CC-FR2-10T, resistant to ultraviolet, ionizing radiation, and chemicals which may cause DNA damage, was identified in Taiwan. The expression level of D. ficus RecA, which has 92% sequence identity with Deinococcus radiodurans (Dr.) RecA, will be upregulated upon UV radiation. Multiple sequence alignment of RecA proteins from bacteria belonging to Escherichia coli and the Deinococcus genus reveals that the C-terminal tail of D. ficus RecA is shorter and contains less acidic residues than E. coli RecA. D. ficus RecA exhibits a higher ATPase activity toward single-stranded (ss) DNA and efficiently promotes DNA strand exchange that a filament is first formed on ssDNA, followed by uptake of the double-stranded (ds) substrate. Moreover, D. ficus RecA exhibits a pH-reaction profile for DNA strand exchange similar to E. coli ΔC17 RecA. Later, a chimera D. ficus C17 E. coli RecA with more acidic residues in the C-terminal tail was constructed and purified. Increased negativity in the C-terminal tail makes the pH reaction profile for Chimera D. ficus C17 E. coli RecA DNA strand exchange exhibit a reaction optimum similar to E. coli RecA. To sum up, D. ficus RecA exhibits reaction properties in substrate-dependent ATPase activity and DNA strand exchange similar to E. coli RecA. Our data indicate that the negativity in the C-terminal tail plays an important role in the regulation of pH-dependent DNA strand exchange activity.
RESUMO
Eukaryotes have evolved a specific strategy to package DNA. The nucleosome is a 147-base-pair DNA segment wrapped around histone core proteins that plays important roles regulating DNA-dependent biosynthesis and gene expression. Chromatin remodeling complexes (RSC, Remodel the Structure of Chromatin) hydrolyze ATP to perturb DNA-histone contacts, leading to nucleosome sliding and ejection. Here, we utilized tethered particle motion (TPM) experiments to investigate the mechanism of RSC-mediated nucleosome remodeling in detail. We observed ATP-dependent RSC-mediated DNA looping and nucleosome ejection along individual mononucleosomes and dinucleosomes. We found that nucleosome assembly protein 1 (Nap1) enhanced RSC-mediated nucleosome ejection in a two-step disassembly manner from dinucleosomes but not from mononucleosomes. Based on this work, we provide an entire reaction scheme for the RSC-mediated nucleosome remodeling process that includes DNA looping, nucleosome ejection, the influence of adjacent nucleosomes, and the coordinated action between Nap1 and RSC.