Rossi Silvia Emma, Ajazi Arta, Carotenuto Walter, Foiani Marco, Giannattasio Michele
IFOM (Fondazione Istituto FIRC di Oncologia Molecolare), Via Adamello 16, 20139 Milan, Italy; Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.
IFOM (Fondazione Istituto FIRC di Oncologia Molecolare), Via Adamello 16, 20139 Milan, Italy.
Cell Rep. 2015 Oct 6;13(1):80-92. doi: 10.1016/j.celrep.2015.08.073. Epub 2015 Sep 24.
Replication stress activates the Mec1(ATR) and Rad53 kinases. Rad53 phosphorylates nuclear pores to counteract gene gating, thus preventing aberrant transitions at forks approaching transcribed genes. Here, we show that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites, are detrimental in rad53 mutants experiencing replication stress. Rrm3 and Pif1 ablations rescue cell lethality, chromosome fragmentation, replisome-fork dissociation, fork reversal, and processing in rad53 cells. Through phosphorylation, Rad53 regulates Rrm3 and Pif1; phospho-mimicking rrm3 mutants ameliorate rad53 phenotypes following replication stress without affecting replication across pausing elements under normal conditions. Hence, the Mec1-Rad53 axis protects fork stability by regulating nuclear pores and DNA helicases. We propose that following replication stress, forks stall in an asymmetric conformation by inhibiting Rrm3 and Pif1, thus impeding lagging strand extension and preventing fork reversal; conversely, under unperturbed conditions, the peculiar conformation of forks encountering pausing sites would depend on active Rrm3 and Pif1.
复制应激激活Mec1（ATR）和Rad53激酶。Rad53使核孔磷酸化以对抗基因门控，从而防止在接近转录基因的叉处发生异常转变。在此，我们表明，Rrm3和Pif1这两种协助叉跨越暂停位点前进的DNA解旋酶，在经历复制应激的rad53突变体中是有害的。Rrm3和Pif1的缺失挽救了rad53细胞中的细胞致死率、染色体片段化、复制体-叉解离、叉逆转和加工过程。通过磷酸化，Rad53调节Rrm3和Pif1；模拟磷酸化的rrm3突变体在复制应激后改善了rad53表型，而在正常条件下不影响跨越暂停元件的复制。因此，Mec1-Rad53轴通过调节核孔和DNA解旋酶来保护叉的稳定性。我们提出，在复制应激后，叉通过抑制Rrm3和Pif1以不对称构象停滞，从而阻碍滞后链延伸并防止叉逆转；相反，在未受干扰的条件下，遇到暂停位点的叉的特殊构象将取决于活跃的Rrm3和Pif1。
