Dissecting PCNA function with a systematically designed mutation library in yeast

Abstract

AbstractProliferating cell nuclear antigen (PCNA), encoded by POL30 in Saccharomyces cerevisiae, is a key component of DNA metabolism. Here a library consisting of 308 PCNA mutants was designed and synthesized to probe the contribution of each residue to its biological function. Five regions were identified with elevated sensitivity to DNA damaging reagents using high-throughput phenotype screening. Using a series of genetic and biochemical analyses, we demonstrated that one particular mutant, K168A, which displayed severe DNA damage sensitivity, abolished the DNA damage tolerance (DDT) pathway by disrupting interactions between PCNA and Rad5p. Subsequent domain analysis showed that the PCNA/Rad5p interaction is prerequisite for the function of Rad5p in DDT. Our study not only provides a resource in the form of a library of versatile mutants to study PCNA functions, but also reveals a key regulatory function of Rad5p, which highlights the importance of the PCNA-Rad5p interaction.Author summaryPCNA is regarded as the maestro of DNA replication fork because of the astonishing ability to interact with lots of partner proteins that participate in various DNA metabolism processes. However, it has remained elusive as to how does PCNA orchestrate these functions in harmony. Here, we constructed a systematic mutation library of PCNA, which covers every amino acid to map the functional sites of it. This carefully designed synthetic mutant pool could be generally useful and serve as a flexible resource, such as dissecting the functional mechanism of PCNA by genetic relationship analysis with key proteins through Synthetic genetic array. We further dissected the intrinsic mechanism for damage sensitivity of PCNAK168A, the most severe DNA damage sensitive mutant in our alanine scanning mutation library, this helps us to get better understanding of how PCNA participates in DNA damage tolerance (DDT) pathways. Our findings indicate that K168 site is vital for the interaction between DDT related partner proteins and PCNA, and also highlight the importance of the PCNA-Rad5p interaction.