Weimin Zhang
Chenghao Liu
Yicong Lin
Qingyu Wu
Junbiao Dai
aCenter for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China
bShenzhen 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
More InformationCorresponding author: E-mail address: junbiao.dai@siat.ac.cn (Junbiao Dai)
Received Date: 2018-12-22
Accepted Date:2019-03-07
Rev Recd Date:2019-02-27
Available Online: 2019-06-24 Publish Date:2019-06-20
Abstract
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.Keywords: Mutagenesis,
High-throughput,
DNA damage tolerance,
Template switching,
Genetic interaction
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