Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur 784028
Dr. Devyani Haldar
Laboratory of Chromatin Biology and Epigenetics, Centre for DNA Fingerprinting, Hyderabad 500039
DNA is present in a compact form being wrapped on histone proteins. Histone deacetylases (HDACs) are enzymes involved in the removal of acetyl group leading to compaction of the DNA, thereby regulating gene expression. Class III HDACs, the Sirtuins, use NAD+ as cofactor and are involved in gene expression, heterochromatin maintenance, genome stability and replicative life span. Schizosaccharomyces pombe (Fission yeast) has three sirtuins, hst2, sir2 and hst4. Previous reports from our lab have shown that on deletion of Hst4, cells show slow growth, DNA fragmentation and DNA damage sensitivity. Hst4 is a nuclear protein and is known to deacetylate histone 3 Lys 56 and gets degraded on DNA damage treatment. It is a 415 amino acid long protein consisting of: N-Terminal, HDAC(core) and C-Terminal domain. On deleting the N-terminal domain, the protein does not localise into the nucleus leading to DNA fragmentation and the absence of degradation. Further, bioinformatic analysis showed for the presence of 14 amino acid stretch of putative NLS (stretch of positively charged amino acid): RKRKCQSSENASKR site in the N-Terminal domain. On basis of these observations, my first objective was to confirm the putative NLS site. Transformation of NLS-Hst4 clone (construct consisting of the NLS site alone with HDAC core and C-terminal domain) into ROP57 (Δhst4) strain was performed to check for the rescue of localisation by immunofluoroscence, DNA fragmentation by DAPI staining and to perform growth assay. To meet my second objective, that is, to check for degradation of NLS-Hst4 on DNA damage treatment, the NLS-Hst4 already cloned in one vector was subcloned to another vector. Further, the study of degradation by Western blotting and the NLS GFP clone for localisation by confocal microscopy, DNA fragmentation by DAPI staining and growth assay will be carried out in the lab.
Keywords: Sirtuins, gene expression, DNA damage, NLS, degradation