Proliferating cell nuclear antigen restores the enzymatic activity of a DNA ligase I deficient in DNA binding

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Proliferating cell nuclear antigen (PCNA) is crucial for DNA replication and repair, interacting with numerous proteins.
• Family I DNA ligases seal DNA nicks, a critical step in Okazaki fragment maturation and DNA repair.
• The precise interaction surfaces and mechanisms between PCNA and DNA ligase I are not fully understood.

Purpose of the Study:

• To investigate the role of the PCNA-PCNA-interacting peptide (PIP) box interaction in stimulating DNA ligase I activity.
• To elucidate the contribution of different PCNA and DNA ligase I domains to the nick-sealing process.
• To understand how PCNA binding influences DNA ligase I's affinity and catalytic efficiency.

Main Methods:

• Utilized truncated versions of DNA ligase I, including one lacking the PIP box.
• Assessed the nick-sealing activity of DNA ligase I with and without PCNA.
• Investigated the effect of PCNA C-terminal mutants on DNA ligase I activity.
• Measured rate constants for single-turnover ligation reactions.

Main Results:

• PCNA stimulates DNA ligase I activity even when the PIP box is deleted.
• PCNA rescues the activity of DNA ligase I mutants defective in DNA binding.
• PCNA increases DNA ligase I's affinity for nicked DNA substrates, not its catalytic efficiency.
• PCNA C-terminal mutants fail to stimulate *Entamoeba histolytica* DNA ligase I, suggesting a role in allosteric regulation.

Conclusions:

• PCNA's stimulation of DNA ligase I is not solely dependent on the canonical PIP box-interdomain connector loop interaction.
• PCNA enhances DNA ligase I function by improving substrate binding, acting as a scaffold to stabilize protein-DNA interactions.
• The C-terminal region of PCNA may induce conformational changes in DNA ligase I, facilitating its role in DNA repair.