Deficiency of the Arabidopsis Helicase RTEL1 Triggers a SOG1-Dependent Replication Checkpoint in Response to DNA Cross-Links

Abstract

To maintain genome integrity, DNA replication is executed and regulated by a complex molecular network of numerous proteins, including helicases and cell cycle checkpoint regulators. Through a systematic screening for putative replication mutants, we identified an Arabidopsis thaliana homolog of human Regulator of Telomere Length 1 (RTEL1), which functions in DNA replication, DNA repair, and recombination. RTEL1 deficiency retards plant growth, a phenotype including a prolonged S-phase duration and decreased cell proliferation. Genetic analysis revealed that rtel1 mutant plants show activated cell cycle checkpoints, specific sensitivity to DNA cross-linking agents, and increased homologous recombination, but a lack of progressive shortening of telomeres, indicating that RTEL1 functions have only been partially conserved between mammals and plants. Surprisingly, RTEL1 deficiency induces tolerance to the deoxynucleotide-depleting drug hydroxyurea, which could be mimicked by DNA cross-linking agents. This resistance does not rely on the essential replication checkpoint regulator WEE1 but could be blocked by a mutation in the SOG1 transcription factor. Taken together, our data indicate that RTEL1 is required for DNA replication and that its deficiency activates a SOG1-dependent replication checkpoint.

SUV2, which encodes an ATR‐related cell cycle checkpoint and putative plant ATRIP, is required for aluminium‐dependent root growth inhibition in Arabidopsis

“…This suggests that these four factors comprise a still emerging signalling pathway that likely is activated by some unknown deleterious effect of Al on DNA conformation or integrity that negatively affects the replication fork. Such a scenario is supported by the observation that in conjunction with the increase in Al tolerance seen for these mutants, each has an increase in sensitivity to DNA crosslinking agents such as MMC and CDDP, with loss of ATR , ALT2 , or SUV2 resulting in moderate to extreme inhibition to levels of DNA crosslinkers that have little effect on Col‐0 wt (Sakamoto et al ; Nezames et al ; Hu et al ). Consequently, it could be argued that Al is perceived by this ATR‐regulated pathway in a manner that is reminiscent of covalent crosslinkers even though Al apparently does not have the extreme consequences associated with chemicals such as MMC and CDDP.…”

Aluminum-Dependent Terminal Differentiation of the Arabidopsis Root Tip Is Mediated through an ATR-, ALT2-, and SOG1-Regulated Transcriptional Response

“…Such an interaction may cause a conformational change reminiscent of covalent cross-linkers such as Mitomycin C and cisplatin since Al 3+ is expected to have high affinity for the negatively charged phosphodiester DNA backbone (Karlik et al, 1980) and would interact with this backbone differently than divalent cations (Nezames et al, 2012). Interestingly, ATR, ALT2, and SOG1 all respond to DNA cross-linking agents and are linked to Al-dependent stoppage of root growth (Rounds and Larsen, 2008;Nezames et al, 2012;Hu et al, 2015). One could predict that such an interaction would hold DNA in a conformation that negatively impacts replication fork progression, potentially through inhibition of unwinding of genomic DNA since Al 3+ may raise the T m of the double helix (Latha et al, 2002).…”

A flow cytometric method for estimating S-phase duration in plants

“…This technique was first described in mouse epithelium (Quastler and Sherman, 1959) and was the standard method of cell cycle analysis for nearly three decades (Van’t Hof, 1974; Grif et al , 2002; Francis et al , 2008, and references therein). Other methods that have been used to estimate S-phase duration in plants include double labeling (Wimber and Quastler, 1963), cell synchronization (Nagata et al , 1992; Lee et al , 1996; Cools et al , 2010), measurement of cell doubling time (Richard et al , 2001; Menges et al , 2006), kinematic analysis (Dhondt et al , 2010), and analysis of 5-ethynl-2'-deoxyuridine (EdU) labeling kinetics (Hayashi et al , 2013; Hu et al , 2015). …”