The ATR-WEE1 kinase module promotes SUPPRESSOR OF GAMMA RESPONSE 1 translation to activate replication stress responses.

DNA replication stress threatens genome stability and is a hallmark of cancer in humans. The evolutionarily conserved kinases ATR (ATM and RAD3-related) and WEE1 are essential for the activation of replication stress responses. Translational control is an important mechanism that regulates gene expression, but its role in replication stress responses is largely unknown. Here we show that ATR-WEE1 control the translation of SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a master transcription factor required for replication stress responses in Arabidopsis thaliana. Through genetic screening, we found that the loss of GENERAL CONTROL NONDEREPRESSIBLE 20 (GCN20) or GCN1, which function together to inhibit protein translation, suppressed the hypersensitivity of the atr or wee1 mutant to replication stress. Biochemically, WEE1 inhibits GCN20 by phosphorylating it; phosphorylated GCN20 is subsequently polyubiquitinated and degraded. Ribosome profiling experiments revealed that that loss of GCN20 enhanced the translation efficiency of SOG1, while overexpressing GCN20 had the opposite effect. The loss of SOG1 reduced the resistance of wee1 gcn20 to replication stress, whereas overexpressing SOG1 enhanced the resistance to atr or wee1 to replication stress. These results suggest that ATR-WEE1 inhibits GCN20-GCN1 activity to promote the translation of SOG1 during replication stress. These findings link translational control to replication stress responses in Arabidopsis.

A plant-specific module for homologous recombination repair.

Homologous recombination repair (HR) is an error-free DNA damage repair pathway to maintain genome stability and a basis of gene targeting using genome-editing tools. However, the mechanisms of HR in plants are still poorly understood. Through genetic screens for DNA damage response mutants (DDRM) in Arabidopsis, we find that a plant-specific ubiquitin E3 ligase DDRM1 is required for HR. DDRM1 contains an N-terminal BRCT (BRCA1 C-terminal) domain and a C-terminal RING (really interesting new gene) domain and is highly conserved in plants including mosses. The ddrm1 mutant is defective in HR and thus is hypersensitive to DNA-damaging reagents. Biochemical studies reveal that DDRM1 interacts with and ubiquitinates the transcription factor SOG1, a plant-specific master regulator of DNA damage responses. Interestingly, DDRM1-mediated ubiquitination promotes the stability of SOG1. Consistently, genetic data support that SOG1 functions downstream of DDRM1. Our study reveals that DDRM1-SOG1 is a plant-specific module for HR and highlights the importance of ubiquitination in HR.

The multi-BRCT domain protein DDRM2 promotes the recruitment of RAD51 to DNA damage sites to facilitate homologous recombination.

DNA double-strand breaks (DSBs) are the most toxic form of DNA damage in cells. Homologous recombination (HR) is an error-free repair mechanism for DSBs as well as a basis for gene targeting using genome-editing techniques. Despite the importance of HR, the HR mechanism in plants is poorly understood. Through genetic screens for DNA damage response mutants (DDRMs), we find that the Arabidopsis ddrm2 mutant is hypersensitive to DSB-inducing reagents. DDRM2 encodes a protein with four BRCA1 C-terminal (BRCT) domains and is highly conserved in plants including bryophytes, the earliest land plant lineage. The plant-specific transcription factor SOG1 binds to the promoter of DDRM2 and activates its expression. In consistence, the expression of DDRM2 is induced by DSBs in a SOG1-dependent manner. In support, genetic analysis suggests that DDRM2 functions downstream of SOG1. Similar to the sog1 mutant, the ddrm2 mutant shows dramatically reduced HR efficiency. Mechanistically, DDRM2 interacts with the core HR protein RAD51 and is required for the recruitment of RAD51 to DSB sites. Our study reveals that SOG1-DDRM2-RAD51 is a novel module for HR, providing a potential target for improving the efficiency of gene targeting.

The ATR-WEE1 kinase module inhibits the MAC complex to regulate replication stress response.

DNA damage response is a fundamental mechanism to maintain genome stability. The ATR-WEE1 kinase module plays a central role in response to replication stress. Although the ATR-WEE1 pathway has been well studied in yeasts and animals, how ATR-WEE1 functions in plants remains unclear. Through a genetic screen for suppressors of the Arabidopsis atr mutant, we found that loss of function of PRL1, a core subunit of the evolutionarily conserved MAC complex involved in alternative splicing, suppresses the hypersensitivity of atr and wee1 to replication stress. Biochemical studies revealed that WEE1 directly interacts with and phosphorylates PRL1 at Serine 145, which promotes PRL1 ubiquitination and subsequent degradation. In line with the genetic and biochemical data, replication stress induces intron retention of cell cycle genes including CYCD1;1 and CYCD3;1, which is abolished in wee1 but restored in wee1 prl1. Remarkably, co-expressing the coding sequences of CYCD1;1 and CYCD3;1 partially restores the root length and HU response in wee1 prl1. These data suggested that the ATR-WEE1 module inhibits the MAC complex to regulate replication stress responses. Our study discovered PRL1 or the MAC complex as a key downstream regulator of the ATR-WEE1 module and revealed a novel cell cycle control mechanism.

Protein kinase ATR inhibits E3 ubiquitin ligase CRL4PRL1 to stabilize ribonucleotide reductase in response to replication stress.

The protein kinase ATR is essential for replication stress responses in all eukaryotes. Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotide (dNTP), the universal building block for DNA replication and repair. However, the relationship between ATR and RNR is not well understood. Here, we show that ATR promotes the protein stability of RNR in Arabidopsis. Through an activation tagging-based genetic screen, we found that overexpression of TSO2, a small subunit of RNR, partially suppresses the hypersensitivity of the atr mutant to replication stress. Biochemically, TSO2 interacts with PRL1, a central subunit of the Cullin4-based E3 ubiquitin ligase CRL4PRL1, which polyubiquitinates TSO2 and promotes its degradation. ATR inhibits CRL4PRL1 to attenuate TSO2 degradation. Our work provides an important insight into the replication stress responses and a post-translational regulatory mechanism for RNR. Given the evolutionary conservation of the proteins involved, the ATR-PRL1-RNR module may act across eukaryotes.

Salinity regulation of the interaction of halovirus SNJ1 with its host and alteration of the halovirus replication strategy to adapt to the variable ecosystem.

Halovirus is a major force that affects the evolution of extreme halophiles and the biogeochemistry of hypersaline environments. However, until now, the systematic studies on the halovirus ecology and the effects of salt concentration on virus-host systems are lacking. To provide more valuable information for understanding ecological strategies of a virus-host system in the hypersaline ecosystem, we studied the interaction between halovirus SNJ1 and its host Natrinema sp.J7-2 under various NaCl concentrations. We found that the adsorption rate and lytic rate increased with salt concentration, demonstrating that a higher salt concentration promoted viral adsorption and proliferation. Contrary to the lytic rate, the lysogenic rate decreased as the salt concentration increased. Our results also demonstrated that cells incubated at a high salt concentration prior to infection increased the ability of the virus to adsorb and lyse its host cells; therefore, the physiological status of host cells also affected the virus-host interaction. In conclusion, SNJ1 acted as a predator, lysing host cells and releasing progeny viruses in hypersaline environments; in low salt environments, viruses lysogenized host cells to escape the damage from low salinity.