G2/M-checkpoint activation in fasciata1 rescues an aberrant S-phase checkpoint but causes genome instability

Abstract

The WEE1 and ATM AND RAD3-RELATED (ATR) kinases are important regulators of the plant intra-S-phase checkpoint; consequently, WEE1KO and ATRKO roots are hypersensitive to replication-inhibitory drugs. Here, we report on a loss-of function mutant allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex that suppresses the phenotype of WEE1- or ATR-deficient Arabidopsis (Arabidopsis thaliana) plants. We demonstrate that lack of FAS1 activity results in the activation of an ATAXIA TELANGIECTASIA MUTATED (ATM)- and SUPPRESSOR OF GAMMA-RESPONSE 1 (SOG1)-mediated G2/M-arrest that renders the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for the telomere erosion and loss of ribosomal DNA that is described for fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 is an important secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint.

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants

“… 20 ), different levels of histone variants present in rDNA and/or general redistribution of rDNA genes in the nucleolus 15 , 21 . Our analysis of replication foci clustering in the nucleus did not show significant differences between the wild-type and fas1 or nuc1 mutant plants, in line with a recent report suggesting that the S-phase progression is not delayed in the fas1 background 40 .…”

A Mutation in DNA Polymerase α Rescues WEE1KO Sensitivity to HU

“…When plants were treated with 1 mM HU, Col-0 plants showed an extended S-phase duration that is not observed in the wee1-1 plants, consistent with previous reports of the role of WEE1 in arresting the S-phase during the replicative stress response [26,27]. In the polα-2 plants, the HU treatment resulted only in an extended cell cycle duration, whereas in the polα-2 wee1-1 plants both S phase and total cell cycle length increased (Figure 3).…”

“…Studies in other eukaryotes have shown that homologous recombination, and in particular the RAD51 protein, is necessary to stabilize stalled replication forks and, in a later stage, restart them [39]. SMR7 is known to respond to several types of DNA damage, both endogenous [27] and exogenous [28], and is induced to a similar extent in the polymerase ε mutant abo4-1 [40].…”

KU70 and CAF‐1 in Arabidopsis: Divergent roles in rDNA stability and telomere homeostasis

“…Our knowledge so far indicates that the loss of telomeric DNA in fas1 and probably also in fas2 plants is not likely to occur via the same mechanism as of rDNA repeats. Telomere erosion in fas1 is mediated by repair processes activated by ATM, which is supported by the recovery of telomere length to WT levels in fas1 / atm plants (Eekhout et al., 2021). Interestingly though, replicative stress caused by the deficiency of RAD3‐related kinase (ATR) kinase also results in increased inter‐individual heterogeneity of telomeres, as was demonstrated in fas1 / atr plants (Eekhout et al., 2021).…”

“…Apart from histone metabolism, DNA damage processing factors are the major contributors to the telomere and rDNA instability in fas1 . When double‐strand break (DSB) signalling kinase ATAXIA TELANGIECTASIA MUTATED (ATM) is disrupted in fas1 / atm plants, both telomeres and rDNA are protected against the loss (Eekhout et al., 2021). There are two major ways for the cell to repair DSB: non‐homologous end joining (NHEJ) and homologous recombination (HR).…”