Structural Basis for the Interaction Between Yeast Chromatin Assembly Factor 1 and Proliferating Cell Nuclear Antigen

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Proliferating cell nuclear antigen (PCNA) is a homotrimeric protein essential for DNA replication, coordinating protein activities at the replication fork.
• Chromatin assembly factor 1 (CAF-1) is a histone chaperone that deposits histones onto DNA post-replication and interacts with PCNA for nucleosome assembly at silenced regions.
• The precise mechanism by which PCNA discriminates binding partners via short PCNA-interacting peptide (PIP) motifs remains unclear.

Purpose of the Study:

• To elucidate the structural basis of the interaction between the yeast CAF-1 PIP motif and PCNA.
• To investigate the role of flanking amino acids and charge distribution in modulating CAF-1 binding affinity to PCNA.

Main Methods:

• Crystal structure determination of the yeast CAF-1 PIP motif bound to PCNA.
• Utilized a novel strategy for stoichiometric production of the PCNA-PIP complex.
• Performed mutational analysis to assess the impact of flanking amino acid charges on binding affinity.

Main Results:

• The crystal structure revealed that the CAF-1 PIP motif binds to a hydrophobic pocket on PCNA, consistent with known interactions.
• Amino acids flanking the PIP motif interact with the PCNA IDCL or C-terminus, a feature observed in limited other interactions.
• Mutational analysis indicated that positive charges in flanking regions are critical for low micromolar affinity, while upstream negative charges reduce binding.

Conclusions:

• The interaction between CAF-1 and PCNA involves not only the canonical PIP motif but also flanking regions.
• Positive charges in flanking regions are crucial for the specificity and affinity of PCNA-interacting proteins.
• These findings provide insights into how PCNA achieves specificity in recruiting proteins at the replication fork.