Related Experiment Video

Updated: Aug 4, 2026

Optimized PCR-based Detection of Mycoplasma
06:01

Optimized PCR-based Detection of Mycoplasma

Published on: June 20, 2011

Structural and enzymological characterization of the homogeneous deoxyribonucleic acid polymerase from Mycoplasma

L M Boxer, D Korn

    Biochemistry
    |October 16, 1979
    PubMed

    Insights

    Researchers purified the DNA polymerase from Mycoplasma orale, revealing a single, asymmetric protein of 116 kDa. This enzyme lacks exonuclease and endonuclease activity, with moderate processivity in DNA synthesis.

    Area of Science:

    • Molecular Biology
    • Enzymology
    • Microbiology

    Background:

    • Mycoplasma orale is a bacterium known to harbor unique enzymes.
    • Understanding DNA polymerase function is crucial for microbial genetics and DNA replication studies.

    Purpose of the Study:

    • To purify and characterize the DNA polymerase from Mycoplasma orale.
    • To determine the enzyme's structural properties, activity, and processivity.

    Main Methods:

    • Homogeneous purification of Mycoplasma orale DNA polymerase.
    • Sodium dodecyl sulfate (SDS) and two-dimensional gel electrophoresis for protein structure analysis.
    • Ferguson plot analysis, gel filtration, and velocity gradient centrifugation for molecular weight and size determination.
    • Enzyme activity assays to assess specific activity, exonuclease/endonuclease contamination, and polymerization mechanism.

    Main Results:

    • Purified DNA polymerase exhibited a single band of 116,000 daltons on SDS-PAGE, corresponding to polymerase activity.
    • Two-dimensional gel electrophoresis confirmed a single protein species (pI = 6.8) congruent with enzyme activity.
    • Ferguson plot analysis indicated a monomer molecular weight of 140,000, suggesting an asymmetric molecule.
    • The enzyme showed high specific activity (approx. 6 x 10^5 units/mg) and was devoid of exo- and endonuclease activities.
    • Polymerization mechanism demonstrated moderate processivity (14 +/- 4 nucleotides/binding event).

    Conclusions:

    • The Mycoplasma orale DNA polymerase is a homogeneous, asymmetric enzyme of approximately 116 kDa.
    • The purified enzyme is highly active and free from contaminating nuclease activities.
    • Its moderate processivity provides insights into DNA replication mechanisms in Mycoplasma.

    More Related Videos

    Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism11:37

    Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

    Published on: July 28, 2017

    DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis07:38

    DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

    Published on: October 6, 2017

    Related Concept Videos

    Genomic DNA in Prokaryotes00:46

    Genomic DNA in Prokaryotes

    The genome of most prokaryotic organisms consists of double-stranded DNA organized into one circular chromosome in a region of cytoplasm called the nucleoid. The chromosome is tightly wound, or supercoiled, for efficient storage. Prokaryotes also contain other circular pieces of DNA called plasmids. These plasmids are smaller than the chromosome and often carry genes that confer adaptive functions, such as antibiotic resistance.
    Genomic Diversity in Bacteria
    Although bacterial genomes are much...
    Translesion DNA Polymerases02:10

    Translesion DNA Polymerases

    Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
    TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
    Bacterial RNA Polymerase00:43

    Bacterial RNA Polymerase

    Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
    In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
    Eukaryotic RNA Polymerases00:58

    Eukaryotic RNA Polymerases

    RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
    All three eukaryotic RNAPs require specific transcription factors, of which the...
    DNA Isolation01:24

    DNA Isolation

    DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
    Nucleic Acid Structure01:25

    Nucleic Acid Structure

    The pentose sugar in DNA is deoxyribose, while in RNA the pentose sugar is ribose. The difference between the sugars is the presence of the hydroxyl group on the ribose's second carbon and a hydrogen on the deoxyribose's second carbon. The phosphate residue attaches to the hydroxyl group of the 5′ carbon of one sugar and the hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, which forms  a 5′ to 3′ phosphodiester linkage.
    DNA Structure
    DNA has a double-helix structure. The...