Phosphorylation of Ku70 subunit by cell cycle kinases modulates the replication related function of Ku heterodimer.

The Ku protein, a heterodimer of Ku70 and Ku80, binds to chromosomal replication origins maximally at G1-phase and plays an essential role in assembly of origin recognition complex. However, the mechanism regulating such a critical periodic activity of Ku remained unknown. Here, we establish human Ku70 as a novel target of cyclin B1-Cdk1, which phosphorylates it in a Cy-motif dependent manner. Interestingly, cyclin E1- and A2-Cdk2 also phosphorylate Ku70, and as a result, the protein remains in a phosphorylated state during S-M phases of cell cycle. Intriguingly, the phosphorylation of Ku70 by cyclin-Cdks abolishes the interaction of Ku protein with replication origin due to disruption of the dimer. Furthermore, Ku70 is dephosphorylated in G1-phase, when Ku interacts with replication origin maximally. Strikingly, the over-expression of Ku70 with non-phosphorylable Cdk targets enhances the episomal replication of Ors8 origin and induces rereplication in HeLa cells, substantiating a preventive role of Ku phosphorylation in premature and untimely licensing of replication origin. Therefore, periodic phosphorylation of Ku70 by cyclin-Cdks prevents the interaction of Ku with replication origin after initiation events in S-phase and the dephosphorylation at the end of mitosis facilitates its participation in pre-replication complex formation.

DNA Damage and Apoptosis Induction in Cancer Cells by Chemically Engineered Thiolated Riboflavin Gold Nanoassembly.

Herein we have engineered a smart nuclear targeting thiol-modified riboflavin-gold nano assembly, RfS@AuNPs, which accumulates selectively in the nucleus without any nuclear-targeting peptides (NLS/RGD) and shows photophysically in vitro DNA intercalation. A theoretical model using Molecular Dynamics has been developed to probe the mechanism of formation and stability as well as dynamics of the RfS@AuNPs in aqueous solution and within the DNA microenvironment. The RfS@AuNPs facilitate the binucleated cell formation that is reflected in the significant increase of DNA damage marker, γ-H2AX as well as the arrest of most of the HeLa cells at the pre-G1 phase indicating cell death. Moreover, a significant upregulation of apoptotic markers confirms that the cell death occurs through the apoptotic pathway. Analyses of the microarray gene expression of RfS@AuNPs treated HeLa cells show significant alterations in vital biological processes necessary for cell survival. Taken together, our study reports a unique nuclear targeting mechanism through targeting the riboflavin receptors, which are upregulated in cancer cells and induce apoptosis in the targeted cells.

Antibacterial efficacy of acridine derivatives conjugated with gold nanoparticles.

Adsorption of acridine derivatives viz. 9-aminoacridine hydrochloride hydrate (9AA-HCl), acridine yellow (AY), acridine orange (AO), and proflavine (Pro) on citrate stabilized gold nanoparticle surface were studied using different analytical techniques like UV-vis absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and transmission electron microscopy (TEM). The amine moiety of acridine derivative binds strongly to the gold nanoparticles as confirmed by spectroscopic studies. The plasmon band observed for the wine red colloidal gold at 525 nm in the UV-vis spectrum is characteristic of gold nanoparticles. However, with the addition of acridine derivatives the intensity of the absorption band at 525 nm decreases and a new peak emerges at red-end region - a signature of formation of gold-drug complex. The TEM images show the average size of citrate stabilized gold nanoparticles as 15-20 nm, which becomes larger in the presence of various drugs due to aggregation. From the thermogravimetric analyses (TGA) we have measured the number of drug molecules attached per gold nanoparticle (AuNP). These gold nanoparticles are very important as drug delivery vehicles and for clinical applications it is necessary to understand their activity in vivo. The antibacterial efficacy of drugs coated gold nanoparticles were studied against various strains of Gram positive and Gram negative bacteria. Among the four drugs, 9AA-HCl and AO showed antibacterial activity and for both of them the AuNP conjugated drug showed better antibacterial efficacy than the bare drug. Because of the high penetrating power and large surface area of Au(0), a single gold nanoparticle can adsorb multiple drug molecules, hence this total entity acts as a single group against the bacteria.