Katheeja Muhseena N, Das Shankar Prasad, Laha Suparna
Cell Biology and Molecular Genetics Division, Yenepoya Research Centre, Yenepoya Medical College, Yenepoya (Deemed To Be University), University Road, 3rd floor, Academic block, Deralakatte, Mangalore, 575018, India.
Department of Biochemistry, Bose Institute, P1/12 CIT Scheme VII M, 700 054, Kolkata, India.
Cell Div. 2021 Sep 8;16(1):4. doi: 10.1186/s13008-021-00072-x.
The budding yeast protein Chl1p is a nuclear protein required for sister-chromatid cohesion, transcriptional silencing, rDNA recombination, ageing and plays an instrumental role in chromatin remodeling. This helicase is known to preserve genome integrity and spindle length in S-phase. Here we show additional roles of Chl1p at G1/S phase of the cell cycle following DNA damage.
G1 arrested cells when exposed to DNA damage are more sensitive and show bud emergence with faster kinetics in chl1 mutants compared to wild-type cells. Also, more damage to DNA is observed in chl1 cells. The viability falls synergistically in rad24chl1 cells. The regulation of Chl1p on budding kinetics in G1 phase falls in line with Rad9p/Chk1p and shows a synergistic effect with Rad24p/Rad53p. rad9chl1 and chk1chl1 shows similar bud emergence as the single mutants chl1, rad9 and chk1. Whereas rad24chl1 and rad53chl1 shows faster bud emergence compared to the single mutants rad24, rad53 and chl1. In presence of MMS induced damage, synergistic with Rad24p indicates Chl1p's role as a checkpoint at G1/S acting parallel to damage checkpoint pathway. The faster movement of DNA content through G1/S phase and difference in phosphorylation profile of Rad53p in wild type and chl1 cells confirms the checkpoint defect in chl1 mutant cells. Further, we have also confirmed that the checkpoint defect functions in parallel to the damage checkpoint pathway of Rad24p.
Chl1p shows Rad53p independent bud emergence and Rad53p dependent checkpoint activity in presence of damage. This confirms its requirement in two different pathways to maintain the G1/S arrest when cells are exposed to damaging agents. The bud emergence kinetics and DNA segregation were similar to wild type when given the same damage in nocodazole treated chl1 cells which establishes the absence of any role of Chl1p at the G2/M phase. The novelty of this paper lies in revealing the versatile role of Chl1p in checkpoints as well as repair towards regulating G1/S transition. Chl1p thus regulates the G1/S phase by affecting the G1 replication checkpoint pathway and shows an additive effect with Rad24p for Rad53p activation when damaging agents perturb the DNA. Apart from checkpoint activation, it also regulates the budding kinetics as a repair gene.
出芽酵母蛋白Chl1p是一种核蛋白，对于姐妹染色单体黏连、转录沉默、核糖体DNA重组、衰老至关重要，并且在染色质重塑中发挥重要作用。已知这种解旋酶在S期维持基因组完整性和纺锤体长度。在此，我们展示了Chl1p在DNA损伤后细胞周期的G1/S期的其他作用。
与野生型细胞相比，处于G1期停滞的细胞在受到DNA损伤时，chl1突变体更敏感，并且出芽动力学更快。此外，在chl1细胞中观察到更多的DNA损伤。在rad24chl1细胞中，存活率协同下降。Chl1p对G1期出芽动力学的调控与Rad9p/Chk1p一致，并与Rad24p/Rad53p表现出协同效应。rad9chl1和chk1chl1的出芽情况与chl1、rad9和chk1单突变体相似。而rad24chl1和rad53chl1的出芽速度比rad24、rad53和chl1单突变体更快。在甲基磺酸甲酯诱导的损伤存在时，与Rad24p协同表明Chl1p在G1/S期作为一个检查点，与损伤检查点途径平行发挥作用。野生型和chl1细胞中DNA含量通过G1/S期的更快移动以及Rad53p磷酸化谱的差异证实了chl1突变体细胞中的检查点缺陷。此外，我们还证实了该检查点缺陷与Rad24p的损伤检查点途径平行发挥作用。
Chl1p在损伤存在时表现出与Rad53p无关的出芽和与Rad53p相关的检查点活性。这证实了在细胞暴露于损伤剂时，它在两条不同途径中对于维持G1/S期停滞是必需的。在用诺考达唑处理的chl1细胞中给予相同损伤时，出芽动力学和DNA分离与野生型相似，这表明Chl1p在G2/M期没有任何作用。本文的新颖之处在于揭示了Chl1p在检查点以及修复中对调节G1/S期转变的多种作用。因此，Chl1p通过影响G1复制检查点途径来调节G1/S期，并且在损伤剂干扰DNA时，对Rad53p激活与Rad24p表现出累加效应。除了检查点激活外，它还作为一个修复基因调节出芽动力学。
