Collaborative Action of Brca1 and CtIP in Elimination of Covalent Modifications from Double-Strand Breaks to Facilitate Subsequent Break Repair

Abstract

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3′ and 5′ ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3′ single-strand overhang at “clean” DSBs, thus initiating homologous recombination (HR)–dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3′ single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIPS332A/−/− cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP+/−/− cells. Finally, CtIPS332A/−/−BRCA1−/− and CtIP+/−/−BRCA1−/− showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.

Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Repairs DNA Damage Induced by Topoisomerases I and II and Base Alkylation in Vertebrate Cells

“…The results of the present study clarify the involvement of Tdp1 in resolving Top2cc-mediated DNA damage. Finally, because CtIP (RBBP8) has recently emerged as a critical factor for the repair of topoisomerase-DNA complexes (26,27,50), we generated double mutant DT40 cells for Tdp1 and CtIP and investigated the relative contribution of the Tdp1-and CtIPdependent pathways in the cellular responses to Top1-and Top2-targeting drugs as well as methyl methanesulfonate (MMS).…”

“…Recent studies show that CtIP, together with BRCA1, acts in the nuclease-mediated elimination of Top1cc and Top2cc (26,27,50). CtIP S332A/Ϫ/Ϫ DT40 cells, which harbor an alanine substitution at residue Ser-332 of CtIP that abrogates its interaction with BRCA1, are hypersensitive to CPT, etoposide, and MMS but remain proficient in homologous recombination (26). Because this function overlaps with that of Tdp1, we generated double mutant DT40 cells (referred to as CtIP S332A/Ϫ/Ϫ;Tdp1Ϫ/Ϫ) to examine the relationship between Tdp1 and CtIP in different types of DNA repair.…”

CtIP-mediated resection is essential for viability and can operate independently of BRCA1

“…Moreover, in mammalian cells, CtIP-BRCA1 complex formation facilitates removal of 53BP1 binding protein RIF1 from DSB regions, which otherwise blocks resection (Escribano-Díaz et al, 2013). However, the physiological role of S327 phosphorylation has been questioned by the finding that the chicken CtIP-S332A protein can efficiently promote DSB repair by HR, apparently independently of BRCA1 interaction (Nakamura et al, 2010) and by the fact that that knock-in mice homozygous for CtIP-S326A allele are neither tumor prone or HR deficient . Thus, the biological importance of the BRCA1-CtIP interaction remains unclear.…”

“…These conclusions were based on the findings that BRCA1-deficient cells exhibit a mild decrease in IR-induced RPA focus formation (Chen et al, 2008;Escribano-Díaz et al, 2013), that BRCA1 and CtIP inhibit RIF1 end-blocking activity in S/G2 (Escribano-Díaz et al, 2013;Zimmermann et al, 2013), and that cells expressing CtIP-327A are defective in HR (Yun and Hiom, 2009). However, subsequent studies in chicken (Nakamura et al, 2010), frog (Peterson et al, 2011), and mouse cells ; analogous to our CtIPnull cells reconstituted with CtIP-S327A), and direct measurements of gene conversion (Chandramouly et al, 2013), argue that the CtIP-BRCA1 interaction is dispensable for HR. Moreover, our finding that restoration of HR in BRCA1-53BP1-deficient cells and increased resection that occurs in the absence of 53BP1 are CtIP dependent, demonstrates that CtIP-mediated resection is BRCA1 independent.…”

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

“…Alternatively, trapped TOP1 can be removed nucleolytically (21,22). For repair of the 5′-phosphotyrosyl-linked TOP2 adducts, only nucleolytic mechanisms were considered (23,24) until a protein known as "TRAF and TNF receptor-associated protein" (TTRAP) or "ETS1-associated protein II" (EAPII) (25) was found to have such an activity (26); that protein has been renamed "TDP2" (Fig. 2B).…”