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Purification and partial characterization of the principal deoxyribonucleic acid polymerase from Mycoplasmatales
Abstract:
In this report we present the first description of the isolation and partial characterization of the deoxyribonucleic acid (DNA) polymerase activity from two species of Mycoplasmatales, Mycoplasma orale type 1 and M. hyorhinis. We have identified only a single DNA polymerase species in the mycoplasma crude extracts, and the enzymes from the two organisms are very similar in their structural and enzymatic properties. The purified polymerase from each source has a specific activity of greater than 50,000 U/mg of protein, a sedimentation coefficient of 5.6s, and an estimated molecular weight by gel filtration of 130,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the most highly purified M. orale fraction contains a single major protein band of 130,000 daltons, which we believe may represent the polymerase protein. The enzymes are most reactive with gapped (activated) DNA and show a marked preference for this primer template over oligodeoxyribonucleotide-initiated homoribo- or homodeoxyribo-polymers. The most purified preparations are devoid of contaminating endonuclease activity and also appear to lack associated 5' leads to 3'- or 3' leads to 5'-exonuclease activities, as determined by highly sensitive assays. The absence of the 3' leads to 5'-exonuclease is particularly remarkable in that this activity is essentially ubiquitous among the DNA polymerases that have thus far been characterized from procaryotes.
Insights
Researchers isolated and characterized a unique DNA polymerase from Mycoplasma species. This enzyme lacks typical exonuclease activity, distinguishing it from other procaryotic DNA polymerases.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Mycoplasmatales are bacteria lacking cell walls.
- DNA polymerases are crucial enzymes for DNA replication and repair.
- Characterization of novel DNA polymerases can reveal unique biological mechanisms.
Purpose of the Study:
- To isolate and partially characterize the DNA polymerase activity from Mycoplasma orale and Mycoplasma hyorhinis.
- To compare the structural and enzymatic properties of DNA polymerases from these two species.
Main Methods:
- Isolation and purification of DNA polymerase from Mycoplasma species.
- Enzymatic assays to determine activity and substrate preference.
- Gel filtration and SDS-PAGE for molecular weight estimation and purity assessment.
- Assays for associated exonuclease and endonuclease activities.
Main Results:
- A single DNA polymerase species was identified in both Mycoplasma orale and M. hyorhinis.
- The enzymes exhibited similar structural and enzymatic properties, with a specific activity >50,000 U/mg.
- Purified polymerases had a sedimentation coefficient of 5.6s and a molecular weight of 130,000.
- Enzymes showed high reactivity with gapped DNA and lacked detectable endonuclease and 5'→3' or 3'→5' exonuclease activities.
Conclusions:
- Mycoplasma DNA polymerases represent a distinct class, notably lacking the 3'→5' exonuclease activity common in other procaryotes.
- This unique characteristic may have implications for DNA replication and repair mechanisms in these organisms.