Dissecting PCNA function with a systematically designed mutant library in yeast

Qingwen Jiang^{a, b},
Weimin Zhang^{a, b},
Chenghao Liu^{a, #},
Yicong Lin^{a, b},
Qingyu Wu^a,
Junbiao Dai^{a, b
, ,}

a.
Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China
b.
Shenzhen Key Laboratory of Synthetic Genomics and Center for Synthetic Genomics, Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China

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Corresponding author: E-mail address: junbiao.dai@siat.ac.cn (Junbiao Dai)

Current address: Department of Biology, Brandeis University, Massachusetts, 02454, USA.

摘要

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Abstract: Proliferating cell nuclear antigen (PCNA), encoded by POL30 in Saccharomyces cerevisiae, is a key component of DNA metabolism. Here, a library consisting of 304 PCNA mutants was designed and constructed to probe the contribution of each residue to the biological function of PCNA. Five regions with elevated sensitivity to DNA damaging reagents were identified using high-throughput phenotype screening. Using a series of genetic and biochemical analyses, we demonstrated that one particular mutant, K168A, has defects in the DNA damage tolerance (DDT) pathway by disrupting the interaction between PCNA and Rad5. Subsequent domain analysis showed that the PCNA-Rad5 interaction is a prerequisite for the function of Rad5 in DDT. Our study not only provides a resource in the form of a library of versatile mutants to study the functions of PCNA, but also reveals a key residue on PCNA (K168) which highlights the importance of the PCNA-Rad5 interaction in the template switching (TS) pathway.
- Mutagenesis /
- High-throughput /
- DNA damage tolerance /
- Template switching /
- Genetic interaction
Current address: Department of Biology, Brandeis University, Massachusetts, 02454, USA.
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参考文献(59)

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