Interdomain connecting loop and J loop structures determine cross-species compatibility of PCNA

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• Eukaryotic proliferating cell nuclear antigen (PCNA) is crucial for DNA replication and repair.
• Despite conserved structure, PCNA function is often species-specific.
• Understanding cross-species incompatibility is key for molecular studies.

Purpose of the Study:

• Determine the X-ray crystal structure of Neurospora crassa PCNA (NcPCNA).
• Investigate structure-function relationships to explain PCNA cross-species incompatibility.
• Identify specific structural elements responsible for functional differences.

Main Methods:

• X-ray crystallography to determine NcPCNA structure.
• Comparative structural analysis of PCNA from different species.
• Site-directed mutagenesis to create NcPCNA variants.
• Functional assays in Saccharomyces cerevisiae.

Main Results:

• NcPCNA structure revealed significant variations in the interdomain connecting loop (IDCL) and J loop compared to other PCNAs.
• These loop differences affect binding affinities for DNA polymerase delta subunit Pol32 and small-molecule inhibitors.
• Mutants of NcPCNA mimicking Saccharomyces cerevisiae PCNA (ScPCNA) structures were fully functional in yeast.
• Yeast expressing ScPCNA mimics of NcPCNA showed similar genotoxic agent susceptibility as wild-type ScPCNA.

Conclusions:

• The IDCL and J loop structures are critical determinants of PCNA interspecies functional compatibility.
• These specific structural regions, not just overall architecture, dictate functional cross-species compatibility.
• NcPCNA variants with altered IDCL and J loops can restore function in heterologous systems, validating their importance.