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.
Highlights
Several studies have indicated that plant growth and development under stress is largely influenced by the stability and activity of proteins which are induced and involved in environmental stress response
We have utilized three other recombinant purified distinct N-terminal deletion mutants of this protein, lacking the N-terminal nuclear localization signal (NLS) sequence (Del 1), the BRCT module (Del 2) and the Ser/Pro rich (S-P) domain (Del 3) respectively, in the similar set of experiments to understand the relative influence of the N-terminal domains on the conformational stability of the highly conserved C-terminal catalytic core domain (PolX region, Del 3) and the full length protein as a whole (S1 Fig)
Further elimination of the BRCT domain (Del 2) led to an appreciable decrease in the normalized fluorescence intensity (Fig 1E), while the elimination of the additional S-P domain from Del 2 had no further influence on the tryptophan spectrum in Del 3, suggesting that the two tryptophan residues in the BRCT domain had higher contribution to tryptophan fluorescence intensity than the rest of the three tryptophan residues which are located within the C-terminal PolX domain (Del 3) and the S-P region does not contribute to the Biophysical Characterization of Arabidopsis DNA Polymerase λ tryptophan microenvironment of the protein
Summary
Several studies have indicated that plant growth and development under stress is largely influenced by the stability and activity of proteins which are induced and involved in environmental stress response. Among the four different families of DNA polymerases known in mammalian genome [7], family X DNA polymerase members are small, evolutionarily conserved relatively inaccurate enzymes involved in a number of DNA repair processes, including base excision repair (BER) and repair of double-strand breaks (DSBs) [8,9]. Similar to other mammalian family X members, DNA Pol λ is a relatively small, single-subunit enzyme lacking a 3’-5’ proofreading exonuclease activity and is widespread among higher eukaryotes, both in animals and plants [9]. The BRCT domain acts as the site for mediating protein-protein and protein-DNA interactions, while the Ser-Pro rich region has been indicated as the target site for the posttranslational modifications of Pol λ via phosphorylation for regulating the stability of the protein during progression of cell cycle [10]. The catalytic core comprises of an N-terminal 8 kDa domain, unique to family X DNA Pols and a polymerase domain, organized in fingers, palm and the thumb sub-domains that are common to all polymerases
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