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Complementation of Bacillus subtilis polA mutants by DNA polymerase I from Streptococcus pneumoniae
S Martinez1, P Lopez, M Espinosa
1Centro de Investigaciones Biologicas, C.S.I.C., Madrid, Spain.
Abstract:
The polA gene of Streptococcus pneumoniae cloned in the recombinant plasmid pSM22 is expressed in Bacillus subtilis. Extracts of B. subtilis polA mutants containing pSM22 showed 6 times more DNA polymerase activity than extracts of wild-type cells without the plasmid. Complete complementation of the B. subtilis polA5 and polA59 mutations with respect to in vivo resistance to UV irradiation and methyl methanesulfonate was observed when four copies of the pneumococcal polA gene were present in each cell. Ectopic integration of the polA gene together with a cat marker into the chromosome of B. subtilis gave chromosomal insertions containing single and double doses of the pneumococcal polA gene. Correlation with gene dosage was observed for both chloramphenicol acetyltransferase and DNA polymerase activities measured in vitro. Depending on the number of copies of the S. pneumoniae polA gene present, restoration of DNA repair functions in polA mutants of B. subtilis was either partial or complete.
Insights
The Streptococcus pneumoniae polA gene, when expressed in Bacillus subtilis, restored DNA repair functions. Gene dosage correlated with the extent of DNA repair restoration in polA mutants.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- The polA gene encodes DNA polymerase I, crucial for DNA replication and repair.
- Streptococcus pneumoniae polA gene function and its potential in heterologous expression systems are of interest.
- Bacillus subtilis polA mutants exhibit defects in DNA repair and resistance to genotoxic agents.
Purpose of the Study:
- To investigate the expression and functional complementation of the Streptococcus pneumoniae polA gene in Bacillus subtilis.
- To assess the impact of pneumococcal polA gene dosage on DNA polymerase activity and DNA repair in B. subtilis polA mutants.
Main Methods:
- Cloning of the S. pneumoniae polA gene into a recombinant plasmid (pSM22) for expression in B. subtilis.
- Analysis of DNA polymerase activity in B. subtilis polA mutants harboring the recombinant plasmid.
- Evaluation of in vivo resistance to UV irradiation and methyl methanesulfonate.
- Chromosomal integration of the polA gene and assessment of gene dosage effects on enzyme activities.
Main Results:
- Expression of the S. pneumoniae polA gene in B. subtilis resulted in significantly increased DNA polymerase activity in polA mutants.
- Complete complementation of UV and methyl methanesulfonate resistance was achieved with four copies of the pneumococcal polA gene.
- A direct correlation between the gene dosage of the S. pneumoniae polA gene and both DNA polymerase and chloramphenicol acetyltransferase activities was observed.
Conclusions:
- The Streptococcus pneumoniae polA gene is functionally expressed in Bacillus subtilis.
- The pneumococcal polA gene can restore DNA repair functions in B. subtilis polA mutants, with efficacy dependent on gene copy number.
- This study demonstrates the potential for using heterologous gene expression to complement DNA repair deficiencies.