Identification of New Mutations at the PCNA Subunit Interface that Block Translesion Synthesis.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair by interacting with a large number of proteins involved in these processes. Two amino acid substitutions in PCNA, both located at the subunit interface, have previously been shown to block translesion synthesis (TLS), a pathway for bypassing DNA damage during replication. To better understand the role of the subunit interface in TLS, we used random mutagenesis to generate a set of 33 PCNA mutants with substitutions at the subunit interface. We assayed the full set of mutants for viability and sensitivity to ultraviolet (UV) radiation. We then selected a subset of 17 mutants and measured their rates of cell growth, spontaneous mutagenesis, and UV-induced mutagenesis. All except three of these 17 mutants were partially or completely defective in induced mutagenesis, which indicates a partial or complete loss of TLS. These results demonstrate that the integrity of the subunit interface of PCNA is essential for efficient TLS and that even conservative substitutions have the potential to disrupt this process

Random amino acid substitutions of the PCNA subunit interface.

<p>(<b>A</b>) Front view of the structure of PCNA with the location of the subunit interface highlighted. (<b>B</b>) Side view of the structure of PCNA with the subunit interface shown. (<b>C</b>) A schematic of the interface of PCNA is shown with the proper hydrogen bonds indicated. The wild type residues in β strand I₁ are shown in blue with the substitutions generated at each residue listed below each wild type residue. The wild type residues in β strand D₂ are shown in red with the substitutions generated at each residue listed above each wild type residue.</p

UV sensitivity of the PCNA mutants.

<p>Percent survival was plotted as a function of UV dose for strains expressing the β strand I₁ PCNA mutants <b>(A)</b> and the D₂ PCNA mutants <b>(B)</b>. Also included were strains expressing wild type, K164R, E113G, and G178S PCNA. Experiments were repeated in triplicate and average values are reported.</p

Viability and UV sensitivity of all 33 PCNA mutants by the spotting assay.

<p>Yeast cultures were grown expressing either wild type PCNA or mutant PCNA. These strains were spotted at varying concentrations from 10³−10⁸ cells per ml. The cells were stamped without exposure to UV, with exposure to 50 J/m² UV, and with exposure to 100 J/m² UV.</p

Spontaneous and UV-induced mutagenesis of the PCNA mutants.

<p>The number of UV-induced <i>CAN1</i>^<i>S</i> to <i>can1</i>^<i>r</i> mutants was plotted as a function of UV dose for strains expressing the β strand I₁ PCNA mutants <b>(A)</b> and the D₂ PCNA mutants <b>(B)</b>. Also included were strains expressing the wild type, K164R, E113G, and G178S PCNA. The numbers of spontaneous <i>CAN1</i>^<i>S</i> to <i>can1</i>^<i>r</i> mutants are shown for strains expressing the β strand I₁ PCNA mutants <b>(C)</b> and the D₂ PCNA mutants <b>(D)</b>. Experiments were repeated in triplicate and average values are reported.</p