Bellini Angela, Girard Pierre-Marie, Tessier Ludovic, Sage Evelyne, Francesconi Stefania
Institut Curie, Centre de Recherche, Orsay, France; CNRS UMR 3348, Bât. 110, Centre Universitaire, Orsay, France.
PLoS One. 2014 Apr 18;9(4):e95788. doi: 10.1371/journal.pone.0095788. eCollection 2014.
Rad52 is a key protein in homologous recombination (HR), a DNA repair pathway dedicated to double strand breaks and recovery of blocked or collapsed replication forks. Rad52 allows Rad51 loading on single strand DNA, an event required for strand invasion and D-loop formation. In addition, Rad52 functions also in Rad51 independent pathways because of its ability to promote single strand annealing (SSA) that leads to loss of genetic material and to promote D-loops formation that are cleaved by Mus81 endonuclease. We have previously reported that fission yeast Rad52 is phosphorylated in a Sty1 dependent manner upon oxidative stress and in cells where the early step of HR is impaired because of lack of Rad51. Here we show that Rad52 is also constitutively phosphorylated in mus81 null cells and that Sty1 partially impinges on such phosphorylation. As upon oxidative stress, the Rad52 phosphorylation in rad51 and mus81 null cells appears to be independent of Tel1, Rad3 and Cdc2. Most importantly, we show that mutating serine 365 to glycine (S365G) in Rad52 leads to loss of the constitutive Rad52 phosphorylation observed in cells lacking Rad51 and to partial loss of Rad52 phosphorylation in cells lacking Mus81. Contrariwise, phosphorylation of Rad52-S365G protein is not affected upon oxidative stress. These results indicate that different Rad52 residues are phosphorylated in a Sty1 dependent manner in response to these distinct situations. Analysis of spontaneous HR at direct repeats shows that mutating serine 365 leads to an increase in spontaneous deletion-type recombinants issued from mitotic recombination that are Mus81 dependent. In addition, the recombination rate in the rad52-S365G mutant is further increased by hydroxyurea, a drug to which mutant cells are sensitive.
Rad52是同源重组（HR）中的关键蛋白，同源重组是一种致力于双链断裂修复以及受阻或崩溃的复制叉恢复的DNA修复途径。Rad52能使Rad51加载到单链DNA上，这是链入侵和D环形成所必需的事件。此外，Rad52还在不依赖Rad51的途径中发挥作用，因为它能够促进单链退火（SSA），而这会导致遗传物质的丢失，并且能够促进由Mus81核酸内切酶切割的D环形成。我们之前报道过，在氧化应激下以及在由于缺乏Rad51而导致HR早期步骤受损的细胞中，裂殖酵母Rad52会以一种依赖Sty1的方式发生磷酸化。在此我们表明，Rad52在mus81缺失细胞中也会组成性磷酸化，并且Sty1部分影响这种磷酸化。与氧化应激时的情况一样，在rad51和mus81缺失细胞中，Rad52的磷酸化似乎不依赖于Tel1、Rad3和Cdc2。最重要的是，我们表明将Rad52中的丝氨酸365突变为甘氨酸（S365G）会导致在缺乏Rad51的细胞中观察到的组成性Rad52磷酸化丧失，以及在缺乏Mus81的细胞中Rad52磷酸化部分丧失。相反，Rad52 - S365G蛋白的磷酸化在氧化应激时不受影响。这些结果表明，在这些不同情况下，不同的Rad52残基会以依赖Sty1的方式发生磷酸化。对直接重复序列处的自发HR分析表明，丝氨酸365的突变会导致有丝分裂重组产生的自发缺失型重组体增加，这些重组体依赖于Mus81。此外，rad52 - S365G突变体中的重组率会因羟基脲而进一步增加，突变细胞对这种药物敏感。
