Dataset Information

Structural and functional analysis of temperature-sensitive mutants of the phage phi 29 DNA polymerase.

ABSTRACT: The cloning and complete sequencing of gene 2 from four independently isolated temperature-sensitive mutants in the phage phi 29 DNA polymerase (ts2 mutants) is reported. The results obtained indicate that, in vivo, the mutations only affect the initial steps of the replication process. Interestingly, three of these mutations consist in the single amino acid change Ala to Val at position 492 of the protein. The ts2(24) and ts2(98) mutant phi 29 DNA polymerases were expressed, purified and their thermosensitivity was studied at two different steps of DNA replication: 1) protein-primed initiation and 2) elongation of the DNA chain. Whereas the ts2(24) mutation gave rise to a temperature-sensitive phenotype in both reactions, the ts2(98) mutant protein was rather insensitive to the temperature increase. In addition, the ts2(98) mutant protein showed clear differences in the activation by divalent cations. The relationship of these results with structural and functional domains in the phi 29 DNA polymerase are discussed.

SUBMITTER: Blasco MA

PROVIDER: S-EPMC331939 | biostudies-other | 1990 Aug

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Structural and functional analysis of temperature-sensitive mutants of the phage phi 29 DNA polymerase.

Blasco M A MA Blanco L L Parés E E Salas M M Bernad A A

Nucleic acids research 19900801 16

The cloning and complete sequencing of gene 2 from four independently isolated temperature-sensitive mutants in the phage phi 29 DNA polymerase (ts2 mutants) is reported. The results obtained indicate that, in vivo, the mutations only affect the initial steps of the replication process. Interestingly, three of these mutations consist in the single amino acid change Ala to Val at position 492 of the protein. The ts2(24) and ts2(98) mutant phi 29 DNA polymerases were expressed, purified and their ...[more]

PMID: 2118623

Similar Datasets

Project description:The tp53 gene is found to be mutated in 50% of all the cancers. The p53 protein, a product of tp53 gene, is a multi-domain protein. It consists of a core DNA binding domain (DBD) which is responsible for its binding and transcription of downstream target genes. The mutations in p53 protein are responsible for creating cancerous conditions and are found to be occurring at a high frequency in the DBD region of p53. Some of these mutations are also known to be temperature sensitive (ts) in nature. They are known to exhibit partial or strong binding with DNA in the temperature range (298-306 K). Whereas, at 310 K and above they show complete loss in binding. We have analyzed the changes in binding and conformational behavior at 300 K and 310 K for three of the ts-mutants viz., V143A, R249S and R175H. QM-MM simulations have been performed on the wild type and the above mentioned ts-mutants for 30 ns each. The optimal estimate of free energy of binding for a particular number of interface hydrogen bonds was calculated using the maximum likelihood method as described by Chodera et. al (2007). This parameter has been observed to be able to mimic the binding affinity of the p53 ts-mutants at 300 K and 310 K. Thus the correlation between MM-GBSA free energy of binding and hydrogen bonds formed by the interface residues between p53 and DNA has revealed the temperature dependent nature of these mutants. The role of main chain dihedrals was obtained by performing dihedral principal component analysis (PCA). This analysis, suggests that the conformational variations in the main chain dihedrals (? and ?) of the p53 ts-mutants may have caused reduction in the overall stability of the protein. The solvent exposure of the side chains of the interface residues were found to hamper the binding of the p53 to the DNA. Solvent Accessible Surface Area (SASA) also proved to be a crucial property in distinguishing the conformers obtained at 300 K and 310 K for the three ts-mutants from the wild type at 300 K.

Dataset Information

Structural and functional analysis of temperature-sensitive mutants of the phage phi 29 DNA polymerase.

Publications

Structural and functional analysis of temperature-sensitive mutants of the phage phi 29 DNA polymerase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure