Rev7 and 53BP1/Crb2 prevent RecQ helicase-dependent hyper-resection of DNA double-strand breaks

Abstract

Poly(ADP ribose) polymerase inhibitors (PARPi) target cancer cells deficient in homology-directed repair of DNA double-strand breaks (DSBs). In preclinical models, PARPi resistance is tied to altered nucleolytic processing (resection) at the 5’ ends of a DSB. For example, loss of either 53BP1 or Rev7/MAD2L2/FANCV derepresses resection to drive PARPi resistance, although the mechanisms are poorly understood. Long-range resection can be catalyzed by two machineries: the exonuclease Exo1, or the combination of a RecQ helicase and Dna2. Here, we develop a single-cell microscopy assay that allows the distinct phases and machineries of resection to be interrogated simultaneously in living S. pombe cells. Using this assay, we find that the 53BP1 orthologue and Rev7 specifically repress long-range resection through the RecQ helicase-dependent pathway, thereby preventing hyper-resection. These results suggest that ‘rewiring’ of BRCA1-deficient cells to employ an Exo1-independent hyper-resection pathway is a driver of PARPi resistance.

Keep moving and stay in a good shape to find your homologous recombination partner

“…Rad9 53BP1 is also recruited by Dbp11 TopBP1 , forming a complex that restrains Dna2-mediated nucleolytic processing (Granata et al 2010;Pfander and Diffley 2011;Villa et al 2018). This seems conserved in human cells, where TOPBP1 stabilizes 53BP1 to the sites of damage to inhibit DSB resection (Cescutti et al 2010;Zimmermann et al 2013;Chapman et al 2013;Ochs et al 2016;Liu et al 2017) and in S. pombe where the 53BP1 orthologue, Crb2, specifically inhibits the RecQ-helicase-dependent long-range resection pathway (Leland et al 2018). In both S. pombe and mammalian cells, another resection inhibitor, Rev7 seems to be at play although its mechanism of action is unknown (Boersma et al 2015;Xu et al 2015;Leland et al 2018).…”

“…This seems conserved in human cells, where TOPBP1 stabilizes 53BP1 to the sites of damage to inhibit DSB resection (Cescutti et al 2010;Zimmermann et al 2013;Chapman et al 2013;Ochs et al 2016;Liu et al 2017) and in S. pombe where the 53BP1 orthologue, Crb2, specifically inhibits the RecQ-helicase-dependent long-range resection pathway (Leland et al 2018). In both S. pombe and mammalian cells, another resection inhibitor, Rev7 seems to be at play although its mechanism of action is unknown (Boersma et al 2015;Xu et al 2015;Leland et al 2018). On the opposite, Rad9 53BP1 -mediated resection inhibition is counteracted by the Slx4-Rtt107 scaffold that compete for the interaction with Dbp11 and by the Fun30 SMARCAD1 chromatin remodeler Costelloe et al 2012;Eapen et al 2012;Dibitetto et al 2016; reviewed in; Shimada and Gasser 2017).…”

CRL4Cdt2 ubiquitin ligase regulates Dna2 and Rad16 (XPF) nucleases by targeting Pxd1 for degradation

“…In S . pombe , the Dna2- and Rqh1-dependent branch of long-range resection is inhibited by Pxd1 [ 11 ], the 53BP1 ortholog Crb2 [ 82 ], and Rad52 [ 83 ], suggesting that S . pombe cells employ a multi-pronged strategy to inhibit Dna2-mediated resection and consequently restrict SSA.…”

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways

“…While Exo1- and Sgs1-depenent end resection appear redundant in budding yeast [338, 348], in fission yeast wild type cells utilize specifically the Exo1 pathway [349, 350]. Our work suggests that one consequence of up-regulation of the RecQ helicase-mediated end resection pathway is more rapid (and therefore likely more extensive) resection [350]. However, the mechanisms and consequences of resection mechanism choice remain poorly understood.…”