PCNA activates the Holliday junction endonuclease Hjc

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Hjc is a conserved archaeal enzyme that resolves Holliday junctions, crucial structures in homologous recombination and DNA repair.
• Bacterial RuvAB complex mediates Holliday junction binding and branch migration, but archaeal and eukaryotic equivalents remain unidentified.

Purpose of the Study:

• To investigate the interaction between the archaeal Holliday junction resolving enzyme Hjc and the sliding clamp PCNA.
• To determine if PCNA influences Hjc's enzymatic activity.

Main Methods:

• Co-immunoprecipitation assays to detect physical interaction between Hjc and PCNA.
• In vitro enzymatic assays to measure Hjc's Holliday junction cleavage activity in the presence and absence of PCNA.
• Site-directed mutagenesis to delete the PCNA-interacting peptide (PIP) motif in Hjc.

Main Results:

• Hjc from Sulfolobus solfataricus physically interacts with PCNA through a C-terminal PIP motif.
• PCNA significantly stimulates the in vitro Holliday junction cleavage activity of Hjc.
• Deletion of the PIP motif abolished the stimulatory effect of PCNA on Hjc activity.

Conclusions:

• This study reports the first functional interaction between a sliding clamp (PCNA) and a Holliday junction resolving enzyme (Hjc).
• PCNA may act as a scaffold to recruit or modulate the activity of Hjc at Holliday junctions in vivo.
• This interaction opens new avenues for understanding DNA repair mechanisms in archaea and potentially other domains of life.