Identification of αCTD residues important for CI-dependent activation of

<p><b>Copyright information:</b></p><p>Taken from "The C-terminal domain of the RNA polymerase α subunit plays a role in the CI-dependent activation of the bacteriophage λ promoter"</p><p></p><p>Nucleic Acids Research 2007;35(7):2311-2320.</p><p>Published online 27 Mar 2007</p><p>PMCID:PMC1874639.</p><p>© 2007 The Author(s)</p> () Strain WAM106, containing plasmids pTJSpM, pJMH1 and pACλcI, was transformed with each of a set of plasmids encoding the RNAP α subunit in which each residue of αCTD was changed individually to alanine. Cultures were grown at 37°C to OD≈0.2 in LB medium containing appropriate antibiotics, at which time IPTG was added to a final concentration of 0.1 mM. After 60 min induction of α and CI synthesis, the β-galactosidase activities were determined. The activities are presented relative to the activity of the strain harbouring plasmid pLAW2 encoding wild-type α (100% = 2300 Miller units) and are averages of at least three independent experiments. Grey bars indicate positions when alanine occurs naturally. Black bars correspond to the residues in which alanine substitution causes a decrease in activity of ≥20% compared to wild-type α. () Structure of αCTD, showing in black the residues that are important for CI-dependent activation of . Residue K271 is highlighted in grey for reference

S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

<p>The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase. In biochemical assays, we revealed that S100A11 enhanced the RAD51 strand exchange activity. When cells expressing a S100A11 mutant lacking the ability to bind Ca²⁺, a prolonged persistence of RAD51 in repair sites and nuclear γH2AX foci was observed suggesting an incomplete DNA repair. The same phenotype became apparent when S100A11 was depleted by RNA interference. Furthermore, down-regulation of S100A11 resulted in both reduced sister chromatid exchange confirming the restriction of the recombination capacity of the cells, and in an increase of chromosomal aberrations reflecting the functional requirement of S100A11 for the maintenance of genomic stability. Our data indicate that S100A11 is involved in homologous recombination by regulating the appearance of RAD51 in DSB repair sites. This function requires the calcium-binding activity of S100A11.</p