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DNA polymerase alpha-DNA primase from human lymphoblasts.
G Bialek1, H P Nasheuer, H Goetz
1Department of Chemistry, Max-Planck-Institute for Experimental Medicine, Göttingen, F.R.G.
Biochimica Et Biophysica Acta
|December 20, 1988
Summary
Human DNA polymerase alpha-DNA primase complex was purified using immunoaffinity chromatography. This enzyme synthesizes DNA and initiates RNA primers for double-stranded DNA formation.
Area of Science:
- Molecular Biology
- Biochemistry
- Enzymology
Background:
- DNA polymerase alpha-DNA primase is crucial for DNA replication initiation.
- Previous purification protocols existed for calf thymus enzyme.
Purpose of the Study:
- To purify and characterize the human DNA polymerase alpha-DNA primase complex.
- To understand its subunit composition and enzymatic activity.
Main Methods:
- Immunoaffinity purification using a previously developed protocol.
- Analysis of subunit molecular weights and isoelectric points.
- Assay of primase-initiated DNA synthesis using phi X174 DNA.
Main Results:
- Purified complex consisted of four subunits (195/180 kDa alpha, 68 kDa beta, 55/48 kDa gamma/delta).
- Subunit isoelectric points ranged from 5.5 to 8.0.
- Enzyme synthesized double-stranded DNA from single-stranded DNA, forming 3-10 RNA primers (approx. 11 nucleotides).
- DNA polymerase elongation was semi-processive, incorporating 20-50 nucleotides before dissociation.
Conclusions:
- The human DNA polymerase alpha-DNA primase complex has a defined subunit structure.
- The purified enzyme exhibits primase and DNA polymerase activity essential for DNA replication initiation.