Abstract
A primase activity which permits DNA synthesis by yeast DNA polymerase I on a single-stranded circular phi X174 or M13 DNA or on poly(dT)n has been extensively purified by fractionation of a yeast enzyme extract which supports in vitro replication of the yeast 2-microns plasmid DNA (Kojo, H., Greenberg, B. D., and Sugino, A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 7261-7265). Most of this DNA primase activity was separated from DNA polymerase activity, although a small amount remained associated with DNA polymerase I. The primase, active as a monomer, has a molecular weight of about 60,000. The primase synthesizes oligoribonucleotides of discrete size, mainly eight or nine nucleotides, in the presence of single-stranded template DNA and ribonucleoside 5'-triphosphates; it utilizes deoxyribonucleoside 5'-triphosphates as substrate with 10-fold lower efficiency. Product size, chromatographic properties, alpha-amanitin resistance, and molecular weight of the primase activity distinguish it from RNA polymerases I, II, and III. The DNA products synthesized by both primase and DNA polymerase I on a single-stranded DNA template were 200-500 nucleotides long and covalently linked to oligoribonucleotides at their 5'-ends. Addition of yeast single-stranded DNA-binding protein (Arendes, J., Kim, K. C., and Sugino, A. (1983) Proc. Natl. Acad. Sci. U.S. A. 80, 673-677) stimulated the DNA synthesis 2-3-fold.
Highlights
From the $Laboratory of Genetics, National Institute of Environmental Health Sciences, ResearchTriangle Park, North Carolina 27709 and the TDepartment of Molecular and Population Genetics, University of Georgia, Athens, Georgia30602
Most of this DNA primase activity was separated from DNA polymerase activity, a small amount remained associated with DNA polymerase I
We describe the purification and characterization of this yeast DNA primase activity
Summary
Biochemical fractionation .of this activity was yeast DNA polymerase I on a single-stranded circular undertaken to identify and purify yeast DNA replication. The primase synthesizes oligoribonucleotidesof discrete size, mainly eight or nine nucleotides, in the presenocfe single-stranded template DNA and ribonucleoside 5’-triphosphates;it this study an activity has been purified which is absolutely required for a full reconstitution of the in vitro replication. This activity was separable from both DNA and RNA polymerase activities. Activated calf thymus DNA was prepared as previously described [2].
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