Interplay between primase and replication factor C in the hyperthermophilic archaeon Sulfolobus solfataricus

Area of Science:

• Molecular Biology
• Biochemistry
• Archaea Genetics

Background:

• The heterodimeric primase from hyperthermophilic archaeon Sulfolobus solfataricus synthesizes RNA and DNA primers.
• The mechanism coupling primase activity to DNA polymerase extension in this organism remains unclear.
• Replication Factor C (RFC) is a clamp loader complex crucial for DNA replication.

Purpose of the Study:

• To investigate the interaction between Sulfolobus solfataricus primase and RFC.
• To elucidate the role of RFC in regulating primase activity.
• To understand the coordination of primer synthesis and extension in archaea.

Main Methods:

• Yeast two-hybrid assays to detect protein-protein interactions.
• Co-immunoprecipitation assays to confirm interactions in vitro and in cell extracts.
• Deletion analysis to map interaction domains.
• Enzyme activity assays to measure primase and RFC functions.

Main Results:

• The small subunit of RFC interacts with both catalytic and non-catalytic subunits of primase.
• RFC stimulates dinucleotide formation but inhibits primer synthesis by primase, reducing its DNA template affinity.
• Primase stimulates the ATPase activity of RFC.

Conclusions:

• The interaction between primase and RFC modulates the enzymatic activities of both proteins.
• This interaction likely plays a regulatory role in archaeal DNA replication initiation.
• It may be involved in coordinating primer synthesis and its subsequent transfer to DNA polymerase.