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Structural and functional analysis of temperature-sensitive mutants of the phage phi 29 DNA polymerase
M A Blasco1, L Blanco, E Parés
1Centro de Biología Molecular, Universidad Autónoma, Cantoblanco, Madrid, Spain.
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.
Insights
Researchers sequenced gene 2 from temperature-sensitive phage phi 29 DNA polymerase mutants. Mutations affect DNA replication initiation, with one specific mutation showing altered thermosensitivity and cation activation.
Area of Science:
- Molecular Biology
- Virology
- Enzymology
Background:
- Phage phi 29 DNA polymerase is crucial for viral DNA replication.
- Temperature-sensitive mutants are valuable tools for studying enzyme function under varying conditions.
Purpose of the Study:
- To clone and sequence gene 2 from four temperature-sensitive mutants of phage phi 29 DNA polymerase.
- To investigate the functional impact of specific mutations on DNA replication steps.
Main Methods:
- Gene sequencing of four temperature-sensitive mutants.
- Expression and purification of mutant phi 29 DNA polymerases.
- Thermosensitivity assays at initiation and elongation stages of DNA replication.
- Analysis of divalent cation activation.
Main Results:
- Complete sequencing of gene 2 from four ts2 mutants identified mutations affecting initial replication steps.
- Three mutants shared an Ala to Val substitution at position 492.
- The ts2(24) mutation caused temperature sensitivity in both initiation and elongation.
- The ts2(98) mutation exhibited reduced thermosensitivity and altered cation activation.
Conclusions:
- Mutations in phage phi 29 DNA polymerase gene 2 primarily impact the initiation of DNA replication.
- Specific amino acid changes, like Ala492Val, can differentially affect enzyme thermosensitivity and cofactor requirements.
- These findings provide insights into the structure-function relationships of phage phi 29 DNA polymerase domains.