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Enrichment of DNA polymerase III activity in a DNA membrane complex purified from Pneumococcus: the possible
Abstract:
Three DNA polymerase activities, one related to DNA pol III, have been extracted from a DNA membrane complex purified from Streptococcus pneumoniae. DNA pol III was purified 3300-fold, DNA pol II 2800-fold and DNA pol I 1800-fold. Based on inhibition analysis with a drug known to inhibit DNA pol III activity in Gram positive organisms. 6(p-hydroxyphenyl azo) uracil (HpU), 55% of the total DNA polymerase activity is represented by pol III. In contrast, only 3-5% of the total DNA polymerase activity is inhibited by HpU in crude extracts. The purification of the DNA membrane complex from pneumococcus is modified from an earlier procedure (Firshein 1972). The modified procedure results in the separation of three distinct DNA-protein-phospholipid subcomplexes of which the one described above contains most of the radioactivity derived from cells pulsed for a short time with (3H)-thymidine. Proteins are involved in binding DNA in each complex and the conformation of DNA in each complex may be different. All of the subcomplexes contain DNA polymerase activity partially sensitive to HpU. These results provide direct evidence for the structural integrity of a complex that may be involved in DNA replication in vivo.
Insights
Researchers purified three DNA polymerase activities from Streptococcus pneumoniae, identifying DNA pol III as the predominant form. This DNA polymerase complex shows sensitivity to HpU, suggesting its role in DNA replication.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- DNA polymerases are crucial enzymes for DNA replication and repair.
- Streptococcus pneumoniae possesses multiple DNA polymerase activities.
- Understanding DNA polymerase function in bacteria is vital for developing antimicrobial strategies.
Purpose of the Study:
- To purify and characterize DNA polymerase activities from a DNA membrane complex in Streptococcus pneumoniae.
- To investigate the relative abundance and properties of different DNA polymerases.
- To assess the potential role of these polymerases in DNA replication.
Main Methods:
- Purification of a DNA membrane complex from Streptococcus pneumoniae.
- Enzymatic assays to quantify DNA polymerase activities (pol I, pol II, pol III).
- Inhibition analysis using 6-(p-hydroxyphenyl azo) uracil (HpU) to differentiate polymerase activities.
Main Results:
- Three distinct DNA polymerase activities were isolated and purified.
- DNA pol III constituted 55% of the total activity in the purified complex, significantly higher than in crude extracts.
- All isolated subcomplexes exhibited DNA polymerase activity partially sensitive to HpU.
Conclusions:
- The study provides evidence for the structural integrity of a DNA membrane complex involved in DNA replication in Streptococcus pneumoniae.
- DNA pol III is a major DNA polymerase in this bacterium.
- The sensitivity to HpU suggests a specific role for DNA pol III in vivo.