Inner nuclear membrane protein Lem2 facilitates Rad3-mediated checkpoint signaling under replication stress induced by nucleotide depletion in fission yeast

Abstract

DNA replication checkpoint is a highly conserved cellular signaling pathway critical for maintaining genome integrity in eukaryotes. It is activated when DNA replication is perturbed. In Schizosaccharomyces pombe, perturbed replication forks activate the sensor kinase Rad3 (ATR/Mec1), which works cooperatively with mediator Mrc1 and the 9-1-1 checkpoint clamp to phosphorylate the effector kinase Cds1 (CHK2/Rad53). Phosphorylation of Cds1 promotes autoactivation of the kinase. Activated Cds1 diffuses away from the forks and stimulates most of the checkpoint responses under replication stress. Although this signaling pathway has been well understood in fission yeast, how the signaling is initiated and thus regulated remains incompletely understood. Previous studies have shown that deletion of lem2+ sensitizes cells to the inhibitor of ribonucleotide reductase, hydroxyurea. However, the underlying mechanism is still not well understood. This study shows that in the presence of hydroxyurea, Lem2 facilitates Rad3-mediated checkpoint signaling for Cds1 activation. Without Lem2, all known Rad3-dependent phosphorylations critical for replication checkpoint signaling were seriously compromised, which likely causes the aberrant mitosis and drug sensitivity observed in this mutant. Interestingly, the mutant is not very sensitive to DNA damage and the DNA damage checkpoint remains largely intact, suggesting that the main function of Lem2 is to facilitate checkpoint signaling in response to replication stress. Since Lem2 is an inner nuclear membrane protein, these results also suggest that the replication checkpoint may be spatially regulated inside nucleus, a previously unknown mechanism.

Inner nuclear membrane protein Lem2 augments heterochromatin formation in response to nutritional conditions

“…It has been reported that lem2Δ cells show an extremely high frequency of the 'cut' (cell untimely torn) phenotype when treated with hydroxyurea (Hayles et al 2013). Furthermore, Rad3-mediated replication checkpoint is severely compromised in lem2Δ cells (Xu 2016). These observations suggest that S. pombe Lem2 has a regulatory role in checkpoint signaling.…”

“…). Furthermore, Rad3‐mediated replication checkpoint is severely compromised in lem2∆ cells (Xu ). These observations suggest that S. pombe Lem2 has a regulatory role in checkpoint signaling.…”

Lem2 is retained at the nuclear envelope through its interaction with Bqt4 in fission yeast

“…Lem2 is associated with chromatin functions, such as the maintenance of the centromeres and the telomeres (Banday et al., ; Barrales et al., ), genome stability and long‐terminal repeat recombination (Tange et al., ), as well as checkpoint signaling (Hayles et al., ; Xu, ). These Lem2 functions, at least in part, are distinct from those of Man1, as man1Δ mutants show phenotypes that are distinct from those of lem2Δ mutants (Barrales et al., ; Gonzalez et al., ; Tange et al., ).…”

Hydroxyurea Induces Cytokinesis Arrest in Cells Expressing a Mutated Sterol-14α-Demethylase in the Ergosterol Biosynthesis Pathway

“…After extensive crosses with all known mutants that are sensitive to HU, including all checkpoint mutants, this hus (HU sensitive) screen identified several new mutants that are highly sensitive to HU. In this paper, we report the characterization of one of our newly screened mutants that was initially described as hus41 (Xu 2016). This mutant was subsequently renamed as erg11-1 in this study after the identification of the mutation and the mutated gene (see below).…”

“…The resulting hus mutants were crossed and compared with all known checkpoint mutants and the mutants that are sensitive to HU to identify novel hus mutants. Preliminary characterization of one of our screened mutants hus41 (Xu 2016) showed that this mutant contains a novel single nucleotide change in the erg11 gene, which causes a G189D mutation in sterol 14a-demethylase (Figure 1). The hus41 mutant was subsequently renamed as erg11-1 mutant in this study.…”