Abstract
The tumor suppressor BRCA2 protects stalled forks from degradation to maintain genome stability. However, the molecular mechanism(s) whereby unprotected forks are stabilized remains to be fully characterized. Here, we demonstrate that WRN helicase ensures efficient restart and limits excessive degradation of stalled forks in BRCA2-deficient cancer cells. In vitro, WRN ATPase/helicase catalyzes fork restoration and curtails MRE11 nuclease activity on regressed forks. We show that WRN helicase inhibitor traps WRN on chromatin leading to rapid fork stalling and nucleolytic degradation of unprotected forks by MRE11, resulting in MUS81-dependent double-strand breaks, elevated non-homologous end-joining and chromosomal instability. WRN helicase inhibition reduces viability of BRCA2-deficient cells and potentiates cytotoxicity of a poly (ADP)ribose polymerase (PARP) inhibitor. Furthermore, BRCA2-deficient xenograft tumors in mice exhibited increased DNA damage and growth inhibition when treated with WRN helicase inhibitor. This work provides mechanistic insight into stalled fork stabilization by WRN helicase when BRCA2 is deficient.
Highlights
The tumor suppressor BRCA2 protects stalled forks from degradation to maintain genome stability
As compared to empty vector (EV) transfected cells, no significant change in median IdU/CldU ratio (0.6196) was observed in cells expressing a WRN mutant deficient in both helicase and exonuclease activities (WRN-K577M/E84A) (Fig. 1b). In agreement with these results, purified recombinant WRN-WT or WRN-E84A catalyzed fork restoration from a model reversed fork (“chicken-foot”) DNA substrate in vitro whereas WRN-K577M failed to do so (Fig. 1c and d), suggesting fork restoration is supported by WRN ATPase/helicase activity. These results suggest that restoration of intact replications forks by WRN helicase is critical for efficient replication resumption following fork stalling when BRCA2 function is lost
WRN ATPase/helicase efficiently restored a model replication fork from reversed fork state in vitro and rescued fork restart defects caused by BRCA2 loss
Summary
The tumor suppressor BRCA2 protects stalled forks from degradation to maintain genome stability. WRN helicase inhibition reduces viability of BRCA2-deficient cells and potentiates cytotoxicity of a poly (ADP)ribose polymerase (PARP) inhibitor. The duty of fork protection extends beyond BRCA1/2 and solicits the involvement of other stress response proteins including ubiquitinated-PCNA6, RAD51 paralogs[7], FANCD28, FANCA8, FANCJ9, BODL110, CTIP11, Abro[112], WRNIP113, EXD214, RIF115, and RAD5216 These fork stabilizing factors are unique and operate independently or synergistically with BRCA1 and BRCA2 to maintain fork integrity[11,14,17]. Increasing fork instability burden through targeted inhibition of compensatory fork stabilizing factors is a promising therapeutic approach to target human cancers with BRCA2 lossof-function mutations. PARP inhibition exacerbates pathological fork degradation and cell death in BRCA2-deficient cells exposed to the replication inhibitor hydroxyurea (HU)[21]
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