Schizosaccharomyces pombe proliferating cell nuclear antigen mutations affect DNA polymerase delta processivity

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Proliferative cell nuclear antigen (PCNA) is a crucial protein involved in DNA replication and repair.
• Understanding PCNA's structure-function relationship is essential for elucidating DNA replication mechanisms.

Purpose of the Study:

• To investigate the role of conserved PCNA residues in fission yeast (Schizosaccharomyces pombe) DNA replication.
• To determine the impact of specific mutations on PCNA's interaction with DNA polymerase delta and replication factor C.

Main Methods:

• Site-directed mutagenesis was used to introduce nine mutations into seven conserved PCNA residues.
• Recombinant wild-type and mutant PCNAs were expressed and purified.
• In vitro assays assessed PCNA's ability to stimulate DNA polymerase delta and replication factor C.
• In vivo studies utilized a null pcna strain (Deltapcna) to evaluate mutant PCNA function and cell phenotypes.

Main Results:

• Mutations in Asp63, Gln201, Glu259, or Glu260 did not significantly impair PCNA function in stimulating DNA synthesis or enhancing polymerase delta processivity.
• Mutations in Leu2 and Arg64 reduced PCNA's capacity to enhance DNA polymerase delta processivity but did not affect ATPase activity of replication factor C.
• Fission yeast strains expressing Leu2 and Arg64 mutant PCNAs exhibited moderate growth defects and elongated phenotypes but were not sensitive to UV irradiation.

Conclusions:

• The side chains of Leu2 and Arg64 on the PCNA trimer ring are critical for tethering DNA polymerase delta, facilitating processive DNA synthesis.
• These residues play a specific role in DNA replication fidelity and efficiency.
• The study provides insights into the molecular mechanisms of PCNA-mediated DNA replication.