Crystal structure of the human primase

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• DNA primase is a DNA-dependent RNA polymerase vital for initiating DNA replication in all organisms.
• Eukaryotic primases are heterodimers comprising catalytic and accessory subunits, both essential for RNA primer synthesis.
• Current understanding of eukaryotic RNA synthesis priming is limited by the absence of full-length primase crystal structures.

Purpose of the Study:

• To determine the crystal structure of full-length human DNA primase.
• To elucidate the enzyme's organization, domain interactions, and substrate binding modes.
• To understand the structural basis for conformational changes during RNA primer synthesis.

Main Methods:

• X-ray crystallography of full-length human primase.
• Modeling of DNA and RNA substrates interacting with the primase structure.

Main Results:

• The crystal structure of full-length human primase was determined, revealing its overall organization and domain positioning.
• The structure illustrates how conformational changes are critical for RNA primer initiation and elongation.
• A long linker between the N- and C-terminal domains of the p58 subunit provides significant conformational flexibility.
• Deletion of this linker impaired both initiation and elongation steps of primer synthesis.

Conclusions:

• The determined structure provides unprecedented insight into human DNA primase function.
• Conformational flexibility, facilitated by the p58 subunit's linker, is essential for primase activity.
• This structural information is key to understanding the regulation of DNA replication initiation.