The DNA damage checkpoint, the first pathway known to be activated
The DNA damage checkpoint, the first pathway known to be activated in response to DNA damage, is a mechanism by which the cell cycle is temporarily arrested to allow DNA repair. years, the extent and the intricacies of the network comprising the G2-M checkpoint still remain obscure, and fundamental questions remain. For instance, the exact mechanism by which DNA damage activates ATR remains elusive. Comprehensive identification of relevant genes will allow a more complete picture of buy 414910-27-3 the system and aid in understanding the underlying molecular mechanisms. Thus, we performed a genome-wide RNA interference (RNAi) screen in cells and confirmed several of the identified genes in vivo. Our results illustrate that checkpoint activation involves the coordinated actions of proteins involved in DNA repair, DNA replication, cell cycle control, chromatin regulation, and RNA processing. Two of the genes, and S2R+ cells. S2R+ cells have an ATR-dependent G2-M checkpoint that can be activated by various DNA-damaging drugs that induce DSBs (7) (fig. S1). For the screen, we used the anticancer drug doxorubicin, which we found brought on the G2-M checkpoint in S2R+ cells. When wild-type cells were treated with doxorubicin, they arrested in G2 phase within 4 hours, as indicated by a decrease in the mitotic index (fig. S1). To identify genes required for this G2 arrest, we first treated cells with individual double-stranded RNAs (dsRNAs) targeting each of the 13,500 genes for 4 days and then treated them with doxorubicin for 4 hours (Fig. 1A). Based on the G2 populace of 30 to 40% (fig. S2) and the population doubling time of 24 hours, we estimated the length of G2 buy 414910-27-3 phase to be roughly 8 hours in S2R+ cells. Our assay, which measured the mitotic index at 4 hours after drug addition, therefore specifically interrogated the requirement for the G2-M checkpoint and not for the G1- or S-phase checkpoints. dsRNAs that abrogated the G2-M checkpoint were identified by the sustained presence of mitotic cells after doxorubicin treatment, which we monitored by phosphorylated histone H3 (pH3) immunostaining. Fig. 1 Genome-wide RNAi screen for genes required for the G2-M checkpoint. (A) Diagram of the assay. S2R+ cells were plated in 384-well plates that contain in each well a dsRNA targeting a particular gene. Cells were incubated for 4 days and treated … In the assay optimization phase, we noticed that knockdown of several buy 414910-27-3 positive control genes, including (ortholog of mammalian (dsRNA that alone does not induce a checkpoint defect. Subsequent analysis for validation was performed without the dsRNA buy 414910-27-3 against mutants that exhibit strong G2-M checkpoint defects in vivo: ((((((((induces a detectable checkpoint defect, we tested them in the same assay conditions as the secondary screens, which used a higher concentration of dsRNA than was used in the primary screen. RNAi-mediated depletion of or led to a moderate G2-M checkpoint defect (fig. S4). We added and to the final list of genes, which brings the total quantity of our candidate genes to 64. Doxorubicin is usually a topoisomerase II (TOP2) inhibitor that damages DNA by trapping TOP2 in a TOP2-DNA complex after TOP2 cuts DNA (14). Therefore, doxorubicin does not damage DNA in the absence of TOP2, raising the possibility that some of the candidate genes recognized in our screen were required specifically for the response to doxorubicin. Consistent with this, TOP2 was identified as one of the strongest hits (Fig. buy 414910-27-3 1B). To distinguish between genes involved in general DNA damage responses and those involved in doxorubicin-specific Pdgfa responses, we performed a checkpoint analysis of the candidate genes, using three additional stimuli: etoposide, bleomycin (Bleocin), and x-rays (fig. S4). Etoposide is usually another TOP2 inhibitor that generates DSBs through TOP2, whereas bleomycin and x-rays directly attack DNA to generate DSBs. Even though response to each stimulus was variable, 59 of the 64 genes consistently scored positive for at least three stimuli (Fig. 1B). Notable exclusions had been TOPORS and Best2, a SUMO (little ubiquitin-like modifier) ligase that goals topoisomerase I (Best1) (15), both which showed an exceptionally solid checkpoint defect just with doxorubicin and etoposide (Fig. 1B). Phosphorylation of histone H2Av, a marker for DNA harm, was undetectable after treatment of cells depleted for either Best2 or TOPORS with doxorubicin (fig. S5). As a result, we assume these genes action upstream.