Protect DNA Ends Research Articles

Olaparib combined with CDK12-IN-3 to promote genomic instability and cell death in ovarian cancer.

Large-scale phase III clinical trials of Olaparib have revealed benefits for ovarian cancer patients with BRCA gene mutations or homologous recombination deficiency (HRD). However, fewer than 50% of ovarian cancer patients have both BRCA mutations and HRD. Therefore, improving the effect of Olaparib in HR-proficient patients is of great clinical value. Here, a combination strategy comprising Olaparib and CDK12-IN-3 effectively inhibited the growth of HR-proficient ovarian cancer in cell line, patient-derived organoid (PDO), and mouse xenograft models. Furthermore, the combination strategy induced severe DNA double-strand break (DSB) formation, increased NHEJ activity in the G2 phase, and reduced HR activity in cancer cells. Mechanistically, the combination treatment impaired Ku80 poly(ADP-ribosyl)ation (PARylation) and phosphorylation, resulting in PARP1-Ku80 complex dissociation. After dissociation, Ku80 occupancy at DSBs and the resulting Ku80-primed NHEJ activity were increased. Owing to Ku80-mediated DNA end protection, MRE11 and Rad51 foci formation was inhibited after the combination treatment, suggesting that this treatment suppressed HR activity. Intriguingly, the combination strategy expedited cGAS nuclear relocalization, further suppressing HR and, conversely, increasing genomic instability. Moreover, the inhibitory effect on cell survival persisted after drug withdrawal. These findings provide a rationale for the clinical application of CDK12-IN-3 in combination with Olaparib.

Dynamics of the DYNLL1-MRE11 complex regulate DNA end resection and recruitment of Shieldin to DSBs.

The extent and efficacy of DNA end resection at DNA double-strand breaks (DSB) determine the repair pathway choice. Here we describe how the 53BP1-associated protein DYNLL1 works in tandem with the Shieldin complex to protect DNA ends. DYNLL1 is recruited to DSBs by 53BP1, where it limits end resection by binding and disrupting the MRE11 dimer. The Shieldin complex is recruited to a fraction of 53BP1-positive DSBs hours after DYNLL1, predominantly in G1 cells. Shieldin localization to DSBs depends on MRE11 activity and is regulated by the interaction of DYNLL1 with MRE11. BRCA1-deficient cells rendered resistant to PARP inhibitors by the loss of Shieldin proteins can be resensitized by the constitutive association of DYNLL1 with MRE11. These results define the temporal and functional dynamics of the 53BP1-centric DNA end resection factors in cells.

DNA-PKcs kinase activity orchestrates both end-processing and end-ligation.

Recent structural studies have revealed the atomic details of how the DNA-PKcs kinase protects DNA ends, recruits and activates Artemis endonuclease for end-processing, and phosphorylates itself for end-ligation, revealing the molecular details from end-processing to end-ligation, two critical phases of the non-homologous end-joining (NHEJ) DNA-double strand break repair pathway.

The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination.

DNA double-strand breaks (DSBs) are highly toxic lesions that can be mended via several DNA repair pathways. Multiple factors can influence the choice and the restrictiveness of repair towards a given pathway in order to warrant the maintenance of genome integrity. During V(D)J recombination, RAG-induced DSBs are (almost) exclusively repaired by the non-homologous end-joining (NHEJ) pathway for the benefit of antigen receptor gene diversity. Here, we review the various parameters that constrain repair of RAG-generated DSBs to NHEJ, including the peculiarity of DNA DSB ends generated by the RAG nuclease, the establishment and maintenance of a post-cleavage synaptic complex, and the protection of DNA ends against resection and (micro)homology-directed repair. In this physiological context, we highlight that certain DSBs have limited DNA repair pathway choice options.

The telomeric 5' end nucleotide is regulated in the budding yeast Naumovozyma castellii.

The junction between the double-stranded and single-stranded telomeric DNA (ds–ss junction) is fundamental in the maintenance of the telomeric chromatin, as it directs the assembly of the telomere binding proteins. In budding yeast, multiple Rap1 proteins bind the telomeric dsDNA, while ssDNA repeats are bound by the Cdc13 protein. Here, we aimed to determine, for the first time, the telomeric 5′ end nucleotide in a budding yeast. To this end, we developed a permutation-specific PCR-based method directed towards the regular 8-mer telomeric repeats in Naumovozyma castellii. We find that, in logarithmically growing cells, the 320 ± 30 bp long telomeres mainly terminate in either of two specific 5′ end permutations of the repeat, both corresponding to a terminal adenine nucleotide. Strikingly, two permutations are completely absent at the 5′ end, indicating that not all ds‐ss junction structures would allow the establishment of the protective telomere chromatin cap structure. Using in vitro DNA end protection assays, we determined that binding of Rap1 and Cdc13 around the most abundant ds–ss junction ensures the protection of both 5′ ends and 3′ overhangs from exonucleolytic degradation. Our results provide mechanistic insights into telomere protection, and reveal that Rap1 and Cdc13 have complementary roles.

Evolutionary mode for the functional preservation of fast-evolving Drosophila telomere capping proteins.

DNA end protection is fundamental for the long-term preservation of the genome. In vertebrates the Shelterin protein complex protects telomeric DNA ends, thereby contributing to the maintenance of genome integrity. In the Drosophila genus, this function is thought to be performed by the Terminin complex, an assembly of fast-evolving subunits. Considering that DNA end protection is fundamental for successful genome replication, the accelerated evolution of Terminin subunits is counterintuitive, as conservation is supposed to maintain the assembly and concerted function of the interacting partners. This problem extends over Drosophila telomere biology and provides insight into the evolution of protein assemblies. In order to learn more about the mechanistic details of this phenomenon we have investigated the intra- and interspecies assemblies of Verrocchio and Modigliani, two Terminin subunits using in vitro assays. Based on our results and on homology-based three-dimensional models for Ver and Moi, we conclude that both proteins contain Ob-fold and contribute to the ssDNA binding of the Terminin complex. We propose that the preservation of Ver function is achieved by conservation of specific amino acids responsible for folding or localized in interacting surfaces. We also provide here the first evidence on Moi DNA binding.

LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells

DNA double-strand break (DSB) repair by homologous recombination (HR) is thought to be restricted to the S- and G2- phases of the cell cycle in part due to 53BP1 antagonizing DNA end resection in G1-phase and non-cycling quiescent (G0) cells. Here, we show that LIN37, a component of the DREAM transcriptional repressor, functions in a 53BP1-independent manner to prevent DNA end resection and HR in G0 cells. Loss of LIN37 leads to the expression of HR proteins, including BRCA1, BRCA2, PALB2, and RAD51, and promotes DNA end resection in G0 cells even in the presence of 53BP1. In contrast to 53BP1-deficiency, DNA end resection in LIN37-deficient G0 cells depends on BRCA1 and leads to RAD51 filament formation and HR. LIN37 is not required to protect DNA ends in cycling cells at G1-phase. Thus, LIN37 regulates a novel 53BP1-independent cell phase-specific DNA end protection pathway that functions uniquely in quiescent cells.

The RNF8 and RNF168 Ubiquitin Ligases Regulate Pro- and Anti-Resection Activities at Broken DNA Ends During Non-Homologous End Joining

The RING-type E3 ubiquitin ligases RNF8 and RNF168 recruit DNA damage response (DDR) factors to chromatin flanking DNA double strand breaks (DSBs) including 53BP1, which protects DNA ends from resection during DNA DSB repair by non-homologous end joining (NHEJ). Deficiency of RNF8 or RNF168 does not lead to demonstrable NHEJ defects, but like deficiency of 53BP1, the combined deficiency of XLF and RNF8 or RNF168 leads to diminished NHEJ in lymphocytes arrested in G0/G1 phase. The function of RNF8 in NHEJ depends on its E3 ubiquitin ligase activity. Loss of RNF8 or RNF168 in G0/G1-phase lymphocytes leads to the resection of broken DNA ends, demonstrating that RNF8 and RNF168 function to protect DNA ends from nucleases, pos sibly through the recruitment of 53BP1. However, the loss of 53BP1 leads to more severe resection than the loss of RNF8 or RNF168. Moreover, in 53BP1-deficient cells, the loss of RNF8 or RNF168 leads to diminished DNA end resection. We conclude that RNF8 and RNF168 regulate pathways that both prevent and promote DNA end resection in cells arrested in G0/G1 phase.

Abstract IA-010: Deciphering DNA end resection at DSBs

Abstract Double stranded DNA break (DSB)s are repaired by two major mechanistically distinct pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). A decisive factor in the choice between DSB repair pathways is in the competition between DNA end protection (necessary for NHEJ) and DNA end resection (necessary for HR). As the ‘master regulator’ of DSB repair pathway choice, 53BP1, a chromatin-associated reader of epigenetic marks at DSBs, helps channel DSBs into the NHEJ pathway by restricting BRCA1-dependent end resection. 53BP1 nucleates the assembly of a higher order ensemble that includes the Shieldin complex, CST complex, and DNA polymerase (Pol) a-primase complex. Loss of BRCA1 provides a therapeutic opportunity as these tumors are exquisitely sensitive to inhibitors of poly (ADP-ribose) polymerase (PARP), and are also susceptible to platinum-based drugs. Loss of 53BP1 or any of the 53BP1-interacting proteins restores end resection and causes PARPi resistance in BRCA1-mutant tumors. We recently identified Dynein light chain like protein 1 (DYNLL1) as as anti-resection factor. Loss of DYNLL1 allowed DNA end resection and restored HR, thereby inducing resistance to platinum drugs and PARPi in BRCA1-mutant tumors. At the molecular level, DYNLL1 limited the nucleolytic resection of DNA ends by interacting with the resection machinery. Importantly, purified DYNLL1 interacted with MRE11 and inhibited its exonuclease activity in vitro. Now we observe that 53BP1 loss prevents DYNLL1 recruitment to DSBs and more broadly to chromatin. We hypothesize that 53BP1 inhibits MRE11-mediated DNA end resection by recruiting DYNLL1 and that DYNLL1 functions in parallel of Shieldin-CST to regulate DNA end resection and DSB repair pathway choice. We are investigating the crosstalk between the Shieldin-CST and DYNLL1-MRE11 arms of the 53BP1-dependent anti-DNA end resection machinery. Citation Format: Dipanjan Chowdhury. Deciphering DNA end resection at DSBs [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-010.

Stabilization of chromatin topology safeguards genome integrity.

To safeguard genome integrity in response to DNA double-strand breaks (DSBs), mammalian cells mobilize the neighbouring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes1. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin-CST-POLα complex2. How this pathway reflects and influences the three-dimensional nuclear architecture is not known. Here we use super-resolution microscopy to show that 53BP1 and RIF1 form an autonomous functional module that stabilizes three-dimensional chromatin topology at sites of DNA breakage. This process is initiated by accumulation of 53BP1 at regions of compact chromatin that colocalize with topologicallyassociatingdomain (TAD) sequences, followed by recruitment of RIF1 to the boundaries between such domains. The alternating distribution of 53BP1 and RIF1 stabilizes several neighbouring TAD-sized structures at a single DBS site into an ordered, circular arrangement. Depletion of 53BP1 or RIF1 (but not shieldin) disrupts this arrangement and leads to decompaction of DSB-flanking chromatin, reduction in interchromatin space, aberrant spreading of DNA repair proteins, and hyper-resection of DNA ends. Similar topological distortions are triggered by depletion of cohesin, which suggests that the maintenance of chromatin structure after DNA breakage involves basic mechanisms that shape three-dimensional nuclear organization. As topological stabilization of DSB-flanking chromatin is independent of DNA repair, we propose that, besides providing a structural scaffold to protect DNA ends against aberrant processing, 53BP1 and RIF1 safeguard epigenetic integrity at loci that are disrupted by DNA breakage.

XLF and H2AX function in series to promote replication fork stability.

XRCC4-like factor (XLF) is a non-homologous end joining (NHEJ) DNA double strand break repair protein. However, XLF deficiency leads to phenotypes in mice and humans that are not necessarily consistent with an isolated defect in NHEJ. Here we show that XLF functions during DNA replication. XLF undergoes cell division cycle 7-dependent phosphorylation; associates with the replication factor C complex, a critical component of the replisome; and is found at replication forks. XLF deficiency leads to defects in replication fork progression and an increase in fork reversal. The additional loss of H2AX, which protects DNA ends from resection, leads to a requirement for ATR to prevent an MRE11-dependent loss of newly synthesized DNA and activation of DNA damage response. Moreover, H2ax-/-:Xlf-/- cells exhibit a marked dependence on the ATR kinase for survival. We propose that XLF and H2AX function in series to prevent replication stress induced by the MRE11-dependent resection of regressed arms at reversed replication forks.

Shieldin - the protector of DNA ends.

DNA double-strand breaks are a threat to genome integrity and cell viability. The nucleolytic processing of broken DNA ends plays a central role in dictating the repair processes that will mend these lesions. Usually, DNA end resection promotes repair by homologous recombination, whereas minimally processed ends are repaired by non-homologous end joining. Important in this process is the chromatin-binding protein 53BP1, which inhibits DNA end resection. How 53BP1 shields DNA ends from nucleases has been an enduring mystery. The recent discovery of shieldin, a four-subunit protein complex with single-stranded DNA-binding activity, illuminated a strong candidate for the ultimate effector of 53BP1-dependent end protection. Shieldin consists of REV7, a known 53BP1-pathway component, and three hitherto uncharacterized proteins: C20orf196 (SHLD1), FAM35A (SHLD2), and CTC-534A2.2 (SHLD3). Shieldin promotes many 53BP1-associated activities, such as the protection of DNA ends, non-homologous end joining, and immunoglobulin class switching. This review summarizes the identification of shieldin and the various models of shieldin action and highlights some outstanding questions requiring answers to gain a full molecular understanding of shieldin function.

Mechanisms of PARP inhibitor resistance in cancer and insights into the DNA damage response

Editorial summaryInhibitors of poly(ADP-ribose) polymerase (PARPi) have entered the clinic for the treatment of patients with cancers that lack homology-directed DNA repair, but drug resistance remains a clinical hurdle. Recent advances in the identification of PARPi resistance mechanisms have yielded a better understanding of DNA end protection and the relevance of endogenous poly(ADP-ribose) glycohydrolase, highlighting new vulnerabilities.

The Shieldin Complex Protects DNA Ends for 53BP1-Mediated DNA Repair

Abstract The shieldin complex, comprised of SHLD1, SHLD2, SHLD3, and REV7, is a 53BP1 effector complex.

53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ.

53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination1,2, and reduced fidelity of long-range V(D)J recombination3. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres4, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis5-7. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive8, unlike 53BP1-deficient cells9,10, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin-a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)-as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway.

The shieldin complex mediates 53BP1-dependent DNA repair.

53BP1 is a chromatin-binding protein that regulates DNA double-strand break (DSB) repair by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14–9, the latter also recruiting REV7/MAD2L2 to break sites10,11. How 53BP1-pathway proteins shield DNA ends is unknown but two models best explain their action: in one model, the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, whereas in a second model, 53BP1 recruits effector proteins with end-protection activity. Here we describe the identification of such a 53BP1 effector complex, Shieldin, which includes C20orf196 (SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to DSB sites in a 53BP1- and RIF1-dependent manner and its SHLD2 subunit binds to ssDNA via OB-fold domains analogous to those of RPA1 and POT1. Loss of Shieldin impairs non-homologous end-joining (NHEJ), leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in Shieldin subunit genes also cause resistance to poly(ADP-ribose) polymerase (PARP) inhibition in BRCA1-deficient cells and tumours due to restoration of homologous recombination (HR). Finally, we show that ssDNA binding by SHLD2 is critical for Shieldin function, consistent with a model where Shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

Shepherding DNA ends: Rif1 protects telomeres and chromosome breaks.

Cells have evolved conserved mechanisms to protect DNA ends, such as those at the termini of linear chromosomes, or those at DNA double-strand breaks (DSBs). In eukaryotes, DNA ends at chromosomal termini are packaged into proteinaceous structures called telomeres. Telomeres protect chromosome ends from erosion, inadvertent activation of the cellular DNA damage response (DDR), and telomere fusion. In contrast, cells must respond to damage-induced DNA ends at DSBs by harnessing the DDR to restore chromosome integrity, avoiding genome instability and disease. Intriguingly, Rif1 (Rap1-interacting factor 1) has been implicated in telomere homeostasis as well as DSB repair. The protein was first identified in Saccharomyces cerevisiae as being part of the proteinaceous telosome. In mammals, RIF1 is not associated with intact telomeres, but was found at chromosome breaks, where RIF1 has emerged as a key mediator of pathway choice between the two evolutionary conserved DSB repair pathways of non-homologous end-joining (NHEJ) and homologous recombination (HR). While this functional dichotomy has long been a puzzle, recent findings link yeast Rif1 not only to telomeres, but also to DSB repair, and mechanistic parallels likely exist. In this review, we will provide an overview of the actions of Rif1 at DNA ends and explore how exclusion of end-processing factors might be the underlying principle allowing Rif1 to fulfill diverse biological roles at telomeres and chromosome breaks.

FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5'-DNA end.

The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork.

Homologous recombination-dependent repair of telomeric DSBs in proliferating human cells.

Telomeres prevent chromosome ends from being recognized as double-stranded breaks (DSBs). Meanwhile, G/C-rich repetitive telomeric DNA is susceptible to attack by DNA-damaging agents. How cells balance the need to protect DNA ends and the need to repair DNA lesions in telomeres is unknown. Here we show that telomeric DSBs are efficiently repaired in proliferating cells, but are irreparable in stress-induced and replicatively senescent cells. Using the CRISPR-Cas9 technique, we specifically induce DSBs at telomeric or subtelomeric regions. We find that DSB repair (DSBR) at subtelomeres occurs in an error-prone manner resulting in small deletions, suggestive of NHEJ. However, DSBR in telomeres involves ‘telomere-clustering', 3′-protruding C-rich telomeric ssDNA, and HR between sister-chromatid or interchromosomal telomeres. DSBR in telomeres is suppressed by deletion or inhibition of Rad51. These findings reveal proliferation-dependent DSBR in telomeres and suggest that telomeric HR, which is normally constitutively suppressed, is activated in the context of DSBR.

Rif1: A Conserved Regulator of DNA Replication and Repair Hijacked by Telomeres in Yeasts.

Rap1-interacting factor 1 (Rif1) was originally identified in the budding yeast Saccharomyces cerevisiae as a telomere-binding protein that negatively regulates telomerase-mediated telomere elongation. Although this function is conserved in the distantly related fission yeast Schizosaccharomyces pombe, recent studies, both in yeasts and in metazoans, reveal that Rif1 also functions more globally, both in the temporal control of DNA replication and in DNA repair. Rif1 proteins are large and characterized by N-terminal HEAT repeats, predicted to form an elongated alpha-helical structure. In addition, all Rif1 homologs contain two short motifs, abbreviated RVxF/SILK, that are implicated in recruitment of the PP1 (yeast Glc7) phosphatase. In yeasts the RVxF/SILK domains have been shown to play a role in control of DNA replication initiation, at least in part through targeted de-phosphorylation of proteins in the pre-Replication Complex. In human cells Rif1 is recruited to DNA double-strand breaks through an interaction with 53BP1 where it counteracts DNA resection, thus promoting repair by non-homologous end-joining. This function requires the N-terminal HEAT repeat-containing domain. Interestingly, this domain is also implicated in DNA end protection at un-capped telomeres in yeast. We conclude by discussing the deployment of Rif1 at telomeres in yeasts from both an evolutionary perspective and in light of its recently discovered global functions.