WEE1 kinase protects the stability of stalled DNA replication forks by limiting CDK2 activity

Abstract

Highlights d WEE1 activity restricts nascent DNA degradation at stalled replication forks d WEE1 suppresses CDK2 to counteract fork degradation d DNA2 is the nuclease responsible for excessive fork degradation d Neither CHK1 nor p21 promote fork protection as WEE1 does

The nucleolar protein GNL3 prevents resection of stalled replication forks

“…If this increased resection is a consequence of increased origin firing, other causes of increased replication origin firing should have a similar effect. Inhibition of ATR or WEE1, for example, increase replication origin firing (Beck et al, 2012; Moiseeva et al, 2017; Moiseeva et al, 2019) and WEE1 inhibition increases DNA resection in response to HU (Elbaek et al, 2022). We therefore tested the effect of inhibiting ATR or WEE1 on resection in response to HU by sequentially labelling cells with IdU and CldU and then treating them with HU for 4 hours, as before, but in the presence of an inhibitor of ATR or an inhibitor of WEE1 (Figure 4G, Figure S4E).…”

Temporal phosphoproteomics reveals WEE1-dependent control of 53BP1 pathway

“… 43 , 63 Accordingly, we have previously shown that WEE1 guards against the degradation of nascent DNA at stalled replication forks by DNA2. 15 Thus, WEE1 may promote the protection of stalled forks from DNA2-driven degradation by specifically limiting CDK-driven phosphorylation of 53BP1 and RIF1. Considering the marked impact on 53BP1 recruitment, adavosertib triggers high CDK activity which not only causes DNA damage but also hinders mechanisms that should mitigate it.…”

“…In accordance with previous studies, we use a relatively high concentration of adavosertib, reminiscent of the blood serum concentration in clinical studies. 8 , 15 , 23 , 27 , 28 , 34 Albeit such concentrations have been they routinely used, they increase the risk of convoluting the data with off-target effects. Here, PSSM-based filtering plays a pivotal role as it allowed us to rule out sites that are unlikely CDK substrates based on the surrounding amino acid sequence.…”

“…Loss of WEE1 activity leads to massive origin firing, nucleotide depletion, MUS81/SLX4-mediated chromosome pulverization, and loss of replication fork protection. 8 , 12 , 13 , 14 , 15 …”

Exploring the genetic space of the DNA damage response for cancer therapy through CRISPR‐based screens

“…The WEE1 kinase phosphorylates and inhibits CDK1 and CDK2, thereby exerting control over the intra‐S and G2‐M checkpoints [ 80 , 81 ]. Recently, WEE1 has been described to protect against fork degradation at stalled replication forks [ 82 ]. Cells with higher levels of endogenous replication stress, for instance promoted by high expression of MYC and cyclin E [ 36 ], were shown to be hypersensitive to WEE1 inhibition.…”