Replication factor C interacts with the C-terminal side of proliferating cell nuclear antigen

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Replication Factor C (RF-C) is a vital heteropentameric protein complex essential for DNA replication and repair.
• RF-C acts as a molecular matchmaker, facilitating the loading of Proliferating Cell Nuclear Antigen (PCNA) onto double-stranded DNA.
• This PCNA loading is a prerequisite for DNA elongation by DNA polymerases delta and epsilon.

Purpose of the Study:

• To elucidate the precise mode of interaction between RF-C and the PCNA clamp.
• To identify the specific regions and subunits of RF-C involved in PCNA binding.
• To determine if the RF-C-PCNA interaction is evolutionarily conserved.

Main Methods:

• Utilized modified human PCNA, 32P-labeled at either the C or N terminus.
• Employed a kinase protection assay to assess RF-C's effect on PCNA phosphorylation.
• Investigated interactions using specific domains of the human RF-C large subunit and the RF-C 36 kDa subunit.

Main Results:

• RF-C protected the C-terminal region (C-side) of PCNA from phosphorylation, indicating interaction at this face.
• The PCNA binding region (residues 478-712) of the RF-C large subunit, but not the DNA binding region, showed a similar protection profile.
• The RF-C 36 kDa subunit independently interacted with the PCNA C-side.
• RF-C from Drosophila melanogaster showed conserved interaction patterns with human PCNA.

Conclusions:

• RF-C primarily interacts with the C-side of PCNA, a critical interface for DNA replication.
• Specific subunits and regions within RF-C mediate this interaction.
• The conserved nature of the RF-C-PCNA interaction underscores its fundamental importance in eukaryotic DNA replication machinery.