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A DNA primase from yeast. Purification and partial characterization
Abstract:
A DNA primase activity has been purified from the budding yeast Saccharomyces. The resulting preparation was nearly homogeneous and was devoid of DNA and RNA polymerase activities. The primase activity cofractionated with a Mr 65,000 polypeptide in sedimentation and chromatography procedures, and the native molecular weight of the enzyme corresponded closely to this value suggesting that the primase or an active proteolytic fragment of the protein exists as a monomer. Both heat-denatured calf thymus DNA and poly(dT) could be utilized by the enzyme as templates. Primase exhibited an absolute requirement for divalent cations and for rATP on a poly(dT) template. Although it required the ribonucleotide to initiate primer chains, the enzyme could incorporate the deoxynucleotide into primers. The product of the primase-catalyzed reaction was an oligonucleotide of discrete length (11-13 nucleotides), and oligonucleotides that were apparently dimers of this unit length were also observed. Primers that were synthesized were virtually identical in size in both the presence and absence of dATP incorporation. Although the bulk of DNA primase activity was isolated as a "free" enzyme, a portion of cellular primase activity co-chromatographed with DNA polymerase suggesting an association between these enzymes similar to that found in several higher eukaryotes.
Insights
Researchers purified a nearly homogeneous DNA primase from yeast, finding it functions as a monomer and synthesizes short DNA primers. This enzyme requires ribonucleotides to start primer synthesis but can incorporate deoxynucleotides, with some activity associating with DNA polymerase.
Area of Science:
- Molecular Biology
- Biochemistry
- Yeast Genetics
Background:
- DNA primase is essential for initiating DNA replication by synthesizing RNA primers.
- Understanding DNA primase function in yeast provides insights into eukaryotic DNA replication mechanisms.
Purpose of the Study:
- To purify and characterize DNA primase activity from Saccharomyces cerevisiae.
- To investigate the enzymatic properties and potential associations of yeast DNA primase.
Main Methods:
- Purification of DNA primase using chromatography and sedimentation techniques.
- Enzymatic assays to determine template usage, cofactor requirements, and product synthesis.
- Molecular weight determination via sedimentation and chromatography.
Main Results:
- A nearly homogeneous DNA primase preparation was obtained, devoid of DNA and RNA polymerase activities.
- The primase activity was associated with a 65 kDa polypeptide, suggesting a monomeric enzyme.
- The enzyme utilized denatured DNA and poly(dT) templates, requiring divalent cations and rATP for initiation, and could incorporate deoxynucleotides into primers.
- Primase synthesized oligonucleotides of 11-13 nucleotides, with some association observed between primase and DNA polymerase activities.
Conclusions:
- The purified yeast DNA primase is a distinct enzymatic entity with specific substrate and cofactor requirements.
- The enzyme's monomeric nature and its association with DNA polymerase suggest conserved mechanisms in eukaryotic DNA replication.