A DNA primase from mouse cells. Purification and partial characterization.

Area of Science:

• Molecular Biology
• Enzymology
• Mammalian Cell Biology

Background:

• DNA replication initiation requires RNA primers synthesized by primase.
• Understanding primase structure and function is crucial for comprehending DNA synthesis.

Purpose of the Study:

• To purify and characterize the enzymatic activity responsible for synthesizing DNA primers in mammalian cells.
• To identify the protein components of the DNA primase complex and their stoichiometry.

Main Methods:

• Purification of enzymatic activity from mouse hybridoma cells using multiple chromatography steps.
• Analysis of protein components by SDS-PAGE and glycerol gradient sedimentation.
• Assay of oligoribonucleotide synthesis and DNA synthesis initiation activity.

Main Results:

• Enzymatic activity purified over 10,000-fold, synthesizing RNA primers of 9-10 nucleotides.
• The most purified fraction contained two protein components (56,000 and 46,000 Da) in a 1:1 ratio, consistent with a native enzyme of 5.5 S.
• Activity required rATP and Mg2+, was template-dependent (preferring poly(dIT)), and was inhibited by beta-D-arabinose-CTP.

Conclusions:

• The purified enzyme exhibits properties consistent with mammalian DNA primase, initiating DNA synthesis via RNA primers.
• The enzyme appears to be a complex of two distinct proteins, suggesting a multi-subunit structure for mammalian primase.
• Characterization provides insights into the molecular mechanisms of DNA replication initiation in mammals.