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 CtIP(S332A/-/-) 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, CtIP(S332A/-/-)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.
The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show ...that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40
cells to the Top1 poison SN-38, it does not sensitise nuclease-dead
cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (
= 0.005) and overall survival (OS) (
= 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.
During S-phase replication forks can stall at specific genetic loci. At some loci, the stalling events depend on the replisome components Schizosaccharomyces pombe Swi1 (Saccharomyces cerevisiae ...Tof1) and Swi3 (S. cerevisiae Csm3) as well as factors that bind DNA in a site-specific manner. Using a new genetic screen we identified Mrc1 (S. cerevisiae Mrc1/metazoan Claspin) as a replisome component involved in replication stalling. Mrc1 is known to form a sub-complex with Swi1 and Swi3 within the replisome and is required for the intra-S phase checkpoint activation. This discovery is surprising as several studies show that S. cerevisiae Mrc1 is not required for replication barrier activity. In contrast, we show that deletion of S. pombe mrc1 leads to an approximately three-fold reduction in barrier activity at several barriers and that Mrc1's role in replication fork stalling is independent of its role in checkpoint activation. Instead, S. pombe Mrc1 mediated fork stalling requires the presence of a functional copy of its phylogenetically conserved DNA binding domain. Interestingly, this domain is on the sequence level absent from S. cerevisiae Mrc1. Our study indicates that direct interactions between the eukaryotic replisome and the DNA are important for site-specific replication stalling.
Tyrosyl DNA phosphodiesterase-1 (TDP1) is the gene product mutated in spinocerebellar ataxia with axonal neuropathy1 (SCAN1). SCAN1 is a hereditary ataxia that lacks extra-neurological phenotype, ...pointing to a critical role for TDP1 in the nervous system. Recently, we showed that TDP1 is associated with the DNA single-strand break (SSBR) repair machinery through an interaction with DNA ligase 3α (Lig3α) and that SCAN1 cells are defective in the repair of chromosomal DNA single-strand breaks (SSBs) arising from abortive Topoisomerase 1 (Top1)–DNA intermediates. Here we demonstrate that TDP1 is also required for the repair of SSBs induced by ionizing radiation (IR), though not measurably for IR-induced DNA double-strand breaks (DSBs). In addition, we provide evidence that abortive Top1 cleavage complexes are processed by the proteasome prior to the action of TDP1 in vivo, and we exploit this observation to show that the SSBR defect in SCAN1 following IR reflects, in part at least, the presence of IR-induced protein–DNA cross-links. Finally we show that TDP1 activity at abortive Top1-SSBs is stimulated by XRCC1/Lig3α in vitro. These data expand the type of SSBs processed by TDP1 to include those induced by ionizing radiation, and raise the possibility that TDP1 inhibitors may improve radiotherapy.
Histone acetyltransferases (HATs) and ATP‐dependent chromatin remodeling factors (ADCRs) are involved in selective gene regulation via modulation of local chromatin configuration. Activation of the ...recombination hotspot ade6‐M26 of Schizosaccharomyces pombe is mediated by a cAMP responsive element (CRE)‐like sequence, M26, and a heterodimeric ATF/CREB transcription factor, Atf1·Pcr1. Chromatin remodeling occurs meiotically around M26. We examined the roles of HATs and ADCRs in chromatin remodeling around M26. Histones H3 and H4 around M26 were hyperacetylated in an M26‐ and Atf1‐dependent manner early in meiosis. SpGcn5, the S. pombe homolog of Gcn5p, was required for the majority of histone H3 acetylation around M26 in vivo. Deletion of gcn5+ caused a significant delay in chromatin remodeling but only partial reduction of M26 meiotic recombination frequency. The snf22+ (a Swi2/Snf2‐ADCR homologue) deletion and snf22+gcn5+ double deletion abolished chromatin remodeling and significant reduction of meiotic recombination around M26. These results suggest that HATs and ADCRs cooperatively alter local chromatin structure, as in selective transcription activation, to activate meiotic recombination at M26 in a site‐specific manner.
For a cancer cell to resist treatment with drugs that trap topoisomerases covalently on the DNA, the topoisomerase must be removed. In this study we provide evidence that the
Schizosaccharomyces ...pombe
Rad32
Mre11
nuclease activity is involved in the removal of both Top2 from 5′ DNA ends, as well as Top1 from 3′ ends
in vivo
. A
ctp1
CtIP
deletion is defective for Top2 removal, but over-proficient for Top1 removal, suggesting that Ctp1
CtIP
plays distinct roles in removing topoisomerases from 5′ and 3′ DNA ends. Analysis of separation of function mutants suggests that MRN dependent topoisomerase removal contributes significantly to resistance against topoisomerase-trapping drugs. This study has important implications for our understanding of the role of the MRN complex and CtIP in resistance of cells to a clinically important group of anti-cancer drugs.
In laboratories in which a colony-replicating robot is not available, manual replication provides a good, low-cost alternative for genetic interaction screening using the Pombe Epistasis Mapper (PEM) ...system. The protocol presented here describes the minimum number of steps required to identify genetic interactions. First, a query deletion is introduced to a library of deletion mutants by mating. Through a series of subsequent selection steps, single and double mutants are isolated and analyzed.
In fission yeast, the small, intrinsically disordered protein S-phase delaying protein 1 (Spd1) blocks DNA replication and causes checkpoint activation at least in part, by inhibiting the enzyme ...ribonucleotide reductase, which is responsible for the synthesis of DNA. The CRL4
E3 ubiquitin ligase mediates degradation of Spd1 and the related protein Spd2 at S phase of the cell cycle. We have generated a conditional allele of CRL4
, by expressing the highly unstable substrate-recruiting protein Cdt2 from a repressible promoter. Unlike Spd1, Spd2 does not regulate deoxynucleotide triphosphate (dNTP) pools; yet we find that Spd1 and Spd2 together inhibit DNA replication upon Cdt2 depletion. To directly test whether this block of replication was solely due to insufficient dNTP levels, we established a deoxy-nucleotide salvage pathway in fission yeast by expressing the human nucleoside transporter human equilibrative nucleoside transporter 1 (hENT1) and the Drosophila deoxynucleoside kinase. We present evidence that this salvage pathway is functional, as 2 µM of deoxynucleosides in the culture medium is able to rescue the growth of two different temperature-sensitive alleles controlling ribonucleotide reductase. However, salvage completely failed to rescue S phase delay, checkpoint activation, and damage sensitivity, which was caused by CRL4
inactivation, suggesting that Spd1-in addition to repressing dNTP synthesis-together with Spd2, can inhibit other replication functions. We propose that this inhibition works at the point of the replication clamp proliferating cell nuclear antigen, a co-factor for DNA replication.
For a cancer cell to resist treatment with drugs that trap topoisomerases covalently on the DNA, the topoisomerase must be removed. In this study, we provide evidence that the Schizosaccharomyces ...pombe Rad32(Mre11) nuclease activity is involved in the removal of both Top2 from 5' DNA ends as well as Top1 from 3' ends in vivo. A ctp1(CtIP) deletion is defective for Top2 removal but overproficient for Top1 removal, suggesting that Ctp1(CtIP) plays distinct roles in removing topoisomerases from 5' and 3' DNA ends. Analysis of separation of function mutants suggests that MRN-dependent topoisomerase removal contributes significantly to resistance against topoisomerase-trapping drugs. This study has important implications for our understanding of the role of the MRN complex and CtIP in resistance of cells to a clinically important group of anticancer drugs.
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