Dynamic DNA-bound PCNA complexes co-ordinate Okazaki fragment synthesis, processing and ligation.

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• DNA replication involves the synthesis, processing, and joining of over a million Okazaki fragments in the human genome.
• Proliferating cell nuclear antigen (PCNA) is loaded onto primer-template junctions by replication factor C (RFC) and interacts with key enzymes for Okazaki fragment processing.
• The coordination mechanism among DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1), and DNA ligase I (LigI) interacting with PCNA remains unclear.

Purpose of the Study:

• To elucidate the coordination of enzymatic activities involved in Okazaki fragment processing and joining.
• To identify novel interactions critical for the completion of Okazaki fragment ligation.

Main Methods:

• Investigated protein-protein interactions using in vitro assays.
• Characterized the roles of Pol δ, FEN1, and LigI in complex formation during DNA synthesis and ligation.
• Analyzed the dynamic association of PCNA with processing enzymes.

Main Results:

• A novel, critical interaction between Pol δ and LigI for Okazaki fragment joining in vitro was identified.
• FEN1 and LigI associate with the PCNA-Pol δ complex during gap-filling synthesis, suggesting a multi-enzyme complex.
• PCNA and LigI remain associated with DNA post-ligation, indicating dissociation of Pol δ and FEN1 during 5' end processing.

Conclusions:

• Dynamic PCNA complexes coordinate Okazaki fragment synthesis and processing.
• A terminal structure involving two linked protein rings of PCNA and LigI encircles the ligated DNA, ensuring efficient genome replication.