title: Understanding the Physical and Molecular Basis of Stability of Arabidopsis DNA Pol λ under UV-B and High NaCl Stress.
abstract: Here, we have investigated the physical and molecular basis of stability of Arabidopsis DNA Pol λ, the sole X family DNA polymerase member in plant genome, under UV-B and salinity stress in connection with the function of the N-terminal BRCT (breast cancer-associated C terminus) domain and Ser-Pro rich region in the regulation of the overall structure of this protein. Tryptophan fluorescence studies, fluorescence quenching and Bis-ANS binding experiments using purified recombinant full length Pol λ and its N-terminal deletion forms have revealed UV-B induced conformational change in BRCT domain deficient Pol λ. On the other hand, the highly conserved C-terminal catalytic core PolX domain maintained its tertiary folds under similar condition. Circular dichroism (CD) and fourier transform infrared (FT-IR) spectral studies have indicated appreciable change in the secondary structural elements in UV-B exposed BRCT domain deficient Pol λ. Increased thermodynamic stability of the C-terminal catalytic core domain suggested destabilizing effect of the N-terminal Ser-Pro rich region on the protein structure. Urea-induced equilibrium unfolding studies have revealed increased stability of Pol λ and its N-terminal deletion mutants at high NaCl concentration. In vivo aggregation studies using transient expression systems in Arabidopsis and tobacco indicated possible aggregation of Pol λ lacking the BRCT domain. Immunoprecipitation assays revealed interaction of Pol λ with the eukaryotic molecular chaperone HSP90, suggesting the possibility of regulation of Pol λ stability by HSP90 in plant cell. Overall, our results have provided one of the first comprehensive information on the biophysical characteristics of Pol λ and indicated the importance of both BRCT and Ser-Pro rich modules in regulating the stability of this protein under genotoxic stress in plants.
authors.affiliation: Protein Chemistry laboratory, Department of Chemistry, Bose Institute, Kolkata, West Bengal, India.
authors.forename: Sujit
authors.initials: S
authors.lastname: Roy
authors.affiliation: Protein Chemistry laboratory, Department of Chemistry, Bose Institute, Kolkata, West Bengal, India.
authors.forename: Victor
authors.initials: V
authors.lastname: Banerjee
authors.affiliation: Protein Chemistry laboratory, Department of Chemistry, Bose Institute, Kolkata, West Bengal, India.
authors.forename: Kali Pada
authors.initials: KP
authors.lastname: Das
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics*; D020134:Catalytic Domain / Q000235:genetics; D004249:DNA Damage / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004151:Dipeptides / Q000235:genetics; D018745:Genome, Plant / Q000235:genetics; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D012965:Sodium Chloride / Q000378:metabolism*; D013312:Stress, Physiological / Q000235:genetics; D014466:Ultraviolet Rays / Q000009:adverse effects*
chemical_list: D029681:Arabidopsis Proteins; D018744:DNA, Plant; D004151:Dipeptides; D018841:HSP90 Heat-Shock Proteins; C465983:seryl-proline; D012965:Sodium Chloride; C109534:DNA polymerase X; D004259:DNA-Directed DNA Polymerase