Dun1, a Chk2-related Kinase, Is the Central Regulator of DNA Damage Signaling

Authors

Candice Qiu Xia Yam1,2, David Boy Chia3, Idina Shi1, Hong Hwa Lim1,2 and Uttam Surana 1,3,2,4

1 Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore
2 Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore
3 Biotransformation Innovation PlatformAgency for Science, Technology and Research (A*STAR), Singapore
4 Department of Pharmacology, National University of Singapore, Singapore

Published in Nucleic Acid Research 2020 11: 202 (Online Version)

 

Abstract

Cells are constantly exposed to genotoxic stresses that cause DNA damage. Left unrepaired, such genomic damage can cause genetic instability, a major feature of cancer and genetic diseases. To avoid such adverse fate, cells trigger an evolutionarily conserved cellular response called DNA damage response (DDR). DDR is a network of interacting pathways that efficiently detect the genomic damage, arrest cells’ progression through the cell cycle and initiate repair process. Central to DDR is the checkpoint control pathway which executes cell cycle arrest and inhibits segregation of damaged chromosomes until they are repaired.

During normal cell division cycle, chromosomes are duplicated during S phase and are held together by a protein complex called cohesins. As the mother cell enters the anaphase phase of mitosis, the cohesion complex is removed by Separase so that the duplicated chromosomes can be partitioned equally to the two daughter cells. The cohesins are protected from Separase by another protein called Securin which ensures that cohesins are not prematurely removed. During mitosis, Securin is marked for proteolytic destruction by a E3 ligase APC (anaphase promoting complex). This allows Separase to dissolve cohesins and chromosomes are partitioned into the daughter cells. When chromosomes are damaged, the checkpoint targets the cohesion-Separase-Securin axis to prevent premature partitioning of damaged chromosomes into daughter cells until they are repaired. Chk1 and Rad53, two important regulators of the checkpoint, are thought to be important for preventing proteolytic destruction of Securin, so that it continues to prevent Separase from dissolving cohesion. Dun1 kinase is also required for this regulatory axis but the mechanism of its action is not known.

This study shows that when chromosomes are damaged, Dun1 is critically important in preventing Securin destruction such that the Dun1-deficient cells prematurely degrade Securin, escape cell cycle arrest and partition the damaged chromosomes into the daughter cells. Surprisingly this occurs even in the presence of the active checkpoint regulators Chk1 and Rad53. Interestingly, proteolytic degradation of Securin in Dun1-deficient cells is mediated not by APC (as is the case during the normal cell cycle) but by another class of E3 ligase Rsp5. Thus, during chromosome damage, cells target the same regulatory scheme as in the normal cell cycle but integrate within it a critical control branch, with Dun1 as the central element. Together, our results suggest a control circuit in which Dun1 is the key regulator preventing premature segregation of damaged chromosomes by inhibiting Rsp5-mediated proteolytic destruction of Securin. Thus, Dun1 and Rsp5 emerge as crucial regulators of DNA damage-induced cell cycle arrest.

2020_08 Hong Hwa Fig 1
Figure 1. (A) Cell morphology, the state of mitotic spindle and the nucleus in both DNA damaged DUN1 and dun1Δ. (B) Top: A schematic diagram depicting the use of bimolecular fluorescence complementation (BiFC) to observe Esp1 and Pds1 interaction in vivo. Pds1 was N-terminally tagged with the 1st-half of Venus tag, (N’-half Venus-Pds1), while Esp1 was tagged at the C-terminus with the 2nd-half of Venus tag (Esp1-C’half Venus). Bottom: G1 synchronized DUN1 CHK1, DUN1 chk1Δ, dun1Δ CHK1, dun1Δ chk1Δ strains containing both Venus-tagged PDS1 and ESP1 were released into YEP+raff+gal medium to induce DNA damage by HO expression. (C) A proposed regulatory scheme depicting the critical role of Dun1 kinase in DNA damage signalling pathway.