Repair Pathway Choice Research Articles

Sp1-dependent recruitment of the histone acetylase p300 to DSBs facilitates chromatin remodeling and recruitment of the NHEJ repair factor Ku70

In response to DNA damage, most factors involved in damage recognition and repair are tightly regulated to ensure proper repair pathway choice. Histone acetylation at DNA double strand breaks (DSBs) by p300 histone acetyltransferase (HAT) is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that phosphorylation of Sp1 by ATM increases its interaction with p300 and that Sp1-dependent recruitment of p300 to DSBs is necessary to modify the histones associated with p300 activity and NHEJ repair factor recruitment and repair. p300 is known to acetylate multiple residues on histones H3 and H4 necessary for NHEJ. Acetylation of H3K18 by p300 is associated with the recruitment of the SWI/SNF chromatin remodeling complex and Ku70 to DSBs for NHEJ repair. Depletion of Sp1 results in decreased acetylation of lysines on histones H3 and H4. Specifically, cells depleted of Sp1 display defects in the acetylation of H3K18, resulting in defective SWI/SNF and Ku70 recruitment to DSBs. These results shed light on mechanisms by which chromatin remodelers are regulated to ensure activation of the appropriate DSB repair pathway.

Abstract 2049: Cell-cycle dependent regulation of Sp1 at DSBs promotes repair via NHEJ and sensitizes cells to PARP inhibition

Abstract DNA double-strand breaks (DSBs) are life-threatening lesions that must be repaired to preserve chromosomal integrity. These lesions are repaired by two primary pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). The factors that mediate pathway choice are critical to maintain genomic integrity. While many of these factors, epigenetic changes and cell cycle stages are known, with HR predominating in S and G2, the underlying mechanisms that determine pathway choice are not well understood. Previously, we found that the transcription factor Sp1 is rapidly recruited to DSBs and is necessary for repair. Here, we demonstrate that Sp1 integrates repair pathway choice with cell cycle by promoting NHEJ in G1. Sp1 localizes to DSBs in G1 to recruit the NHEJ repair factors 53BP1 and Ku70. Sp1 is phosphorylated by Cyclin A/cdk2 upon entry into S phase, evicting both Sp1 and 53BP1 from the break site, thereby permitting BRCA1 binding and HR. Mutation of this phosphorylation site (Sp1-S59A) results in persistence of Sp1 and 53BP1 at DSBs in S phase cells, precluding BRCA1 binding and HR. We also demonstrate that Sp1 is sumoylated in S phase in response to damage. This sumoylation in recognized by the E3-ubiquitin ligase, RNF4, and therefore polyubiquitylates Sp1 and targets it for proteasomal degradation. Expression of a sumoylation deficient Sp1 (Sp1-K16R) results in Sp1 and 53BP1 occupancy at DSBs in S phase, inhibiting BRCA1 binding to DSBs and HR. HR proteins are commonly mutated in cancers, resulting in cellular “addiction” to alternative repair pathways. Exploiting this addiction is the basis of synthetic lethality and is applicable to the treatment of many types of malignancies. Similar to BRCA1 deficiency, expression of Sp1-S59A or Sp1-K16R also increases cell sensitivity to PARP inhibition in BRCA1+/+ cells because of the inability to recruit BRCA1. Alternatively, synthetic lethality in BRCA1-/- cells requires 53BP1, whereas loss of 53BP1 confers PARPi resistance in a BRCA1 deficient cell. Consistent with this, depletion of Sp1 also results in PARPi resistance in BRCA1-/- cells. Interestingly, Sp1 is overexpressed in many tumors and is associated with poor prognosis. These data corroborate our finding that Sp1 is required for NHEJ and provide preliminary evidence that Sp1 may be a biomarker for PARPi sensitivity. Citation Format: Michelle Swift, Jane Azizkhan-Clifford. Cell-cycle dependent regulation of Sp1 at DSBs promotes repair via NHEJ and sensitizes cells to PARP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2049.

A CRISPR-based assay for the study of eukaryotic DNA repair onboard the International Space Station.

As we explore beyond Earth, astronauts may be at risk for harmful DNA damage caused by ionizing radiation. Double-strand breaks are a type of DNA damage that can be repaired by two major cellular pathways: non-homologous end joining, during which insertions or deletions may be added at the break site, and homologous recombination, in which the DNA sequence often remains unchanged. Previous work suggests that space conditions may impact the choice of DNA repair pathway, potentially compounding the risks of increased radiation exposure during space travel. However, our understanding of this problem has been limited by technical and safety concerns, which have prevented integral study of the DNA repair process in space. The CRISPR/Cas9 gene editing system offers a model for the safe and targeted generation of double-strand breaks in eukaryotes. Here we describe a CRISPR-based assay for DNA break induction and assessment of double-strand break repair pathway choice entirely in space. As necessary steps in this process, we describe the first successful genetic transformation and CRISPR/Cas9 genome editing in space. These milestones represent a significant expansion of the molecular biology toolkit onboard the International Space Station.

The evolving complexity of DNA damage foci: RNA, condensates and chromatin in DNA double-strand break repair

Formation of biomolecular condensates is increasingly recognized as a mechanism employed by cells to deal with stress and to optimize enzymatic reactions. Recent studies have characterized several DNA repair foci as phase-separated condensates, behaving like liquid droplets. Concomitantly, the apparent importance of long non-coding RNAs and RNA-binding proteins for the repair of double-strand breaks has raised many questions about their exact contribution to the repair process. Here we discuss how RNA molecules can participate in condensate formation and how RNA-binding proteins can act as molecular scaffolds. We furthermore summarize our current knowledge about how properties of condensates can influence the choice of repair pathway (homologous recombination or non-homologous end joining) and identify the open questions in this field of emerging importance.

Role of Histone Methylation in Maintenance of Genome Integrity.

Packaging of the eukaryotic genome with histone and other proteins forms a chromatin structure that regulates the outcome of all DNA mediated processes. The cellular pathways that ensure genomic stability detect and repair DNA damage through mechanisms that are critically dependent upon chromatin structures established by histones and, particularly upon transient histone post-translational modifications. Though subjected to a range of modifications, histone methylation is especially crucial for DNA damage repair, as the methylated histones often form platforms for subsequent repair protein binding at damaged sites. In this review, we highlight and discuss how histone methylation impacts the maintenance of genome integrity through effects related to DNA repair and repair pathway choice.

Impact of Bioflavonoid Exposure on DNA Damage Response and of Non-Homologous End Joining (NHEJ) Pathway Repair Proteins in Stem Cells

DNA double-strand breaks (DSBs) activate DNA damage response signaling pathways and multiple repair pathways depending on cell cycle stage and differentiation state. An array of recurrent, non-random chromosomal translocations are associated with hematologic malignancies, and we are interested in determining the role of specific DNA repair proteins or pathway choice in the etiology of these rearrangements. Classic non-homologous end joining (NHEJ) utilizes LIG4, XRCC4, and XLF during the final steps; LIG4 and XRCC4 deficiency is implicated in both leukemais and lymphomas but the role of XLF deficiency in leukemia etiology is not clear. Activation of alternative NHEJ (Alt-NHEJ) is largely dependent on CtIP that promotes the resection of DNA ends to uncover micro-homologies promoting the formation of large deletions and chromosomal translocations. Studies suggest that loss of efficient activity in one NHEJ repair pathway may promote use of the other more error-prone pathway. The topoisomerase II poison etoposide is a cytotoxic chemotherapeutic agent associated with therapy-related leukemias characterized by translocations involving the MLL gene at 11q23. Leukemias with MLL gene fusions have been detected in newborns indicating that these translocations can occur in utero, and additional evidence from our lab and others suggests maternal exposure to environmental topoisomerase II inhibitors such as bioflavonoids have the potential to promote these events. We are interested in understanding the crosstalk involved in bioflavonoid exposure, DNA damage response signaling, and repair pathway choice as a model system to understand the underlying mechanisms of oncogenic transformation in infant leukemias.Using established in vitro differentiation protocols, we expose embryonic stem (ES) cells and hematopoietic stem cells (HSC) derived from them to a panel of bioflavonoids alone or in combination for one hour at increasing doses ranging from 0μM-200μM based on determined LD50 values. As a control, cells are exposed to etoposide at the same doses. Following exposure, cells recovered for a period of 0-24hrs, followed by protein extraction and Western blotting. Quantitation of increases in total gH2AX confirm DNA damage, presence of Ku70 and XLF to support classical NHEJ, and CtIP to support Alt-NHEJ. Cells exposed to bioflavonoids genistein and quercetin as well as etoposide show elevated γH2AX levels by 4 hrs post-treatment compared to untreated cells. Parallel studies show dose-dependent increase in the presence of γH2AX foci in these cells. These results confirm that although bioflavonoids have pleiotropic effects, they directly promote DSBs in a short period of time post-exposure. Protein levels of Ku70 and XLF in cells exposed to genistein, quercetin, and etoposide at doses of 25, 50, 75 and 100μM are not significantly different from untreated cells. However, CtIP levels are elevated in cells exposed to quercetin compared to all other groups suggesting a specific potential for this bioflavonoid to promote Alt-NHEJ repair and genome instability.To correlate the activity of these DNA repair protein activities with downstream repair products, we use a model cell system in which parental ES cells were engineered to contain two constructs: GFP exons inserted into MLL and AF9 bcr transgene fragments. Aberrant repair resulting in chromosomal translocations are scored by GFP+ fluorescence. In both ES and derived HSC populations from them translocations can be scored following exposure to bioflavonoids and etoposide. Ongoing experiments include downregulation of XLF or CtIP by siRNA in cells prior to exposure to these compounds. Results will be presented on impact of downregulation of these two NHEJ proteins on 1) amount and persistence of γH2AX; 2) protein levels of other DNA repair pathway proteins, and 3) frequency of generating chromosomal translocations.Understanding the integrated activity and role of these proteins in preventing or promoting translocations, particularly in the context of exposure to a wide range of environmental and dietary compounds, will be critical to understanding the potential for specific isoforms or mutants to increase leukemia risk. DisclosuresNo relevant conflicts of interest to declare.

PRMT5 Regulates DNA Repair By Controlling the Alternative Splicing of Epigenetic Factors

PRMT5 is the major type II arginine methyltransferase that symmetrically dimethylates histone and non-histone substrates, including several components of the RNA splicing machinery. PRMT5 is overexpressed in many cancer types and PRMT5 inhibition has recently been shown to be a promising therapeutic approach for these diseases, including lymphoma and leukemia. PRMT5 plays an important role in adult mouse hematopoiesis, as its conditional deletion in the hematopoietic compartment (using an Mx1-Cre based system) leads to rapid, lethal, bone marrow aplasia (F. Liu et al, JCI 2015). To avoid excess toxicity, we have been investigating the myriad of cellular processes regulated by PRMT5, in order to identify combinatorial approaches, that can exploit potential synthetic vulnerabilities, similar to what has been shown for loss of MTAP and PRMT5 inhibition. Here we report that PRMT5 deletion or inhibition leads to the accumulation of DNA damage, at least in part due to defects in the homologous recombination (HR) DNA repair pathway, which ultimately activates the p53 pathway and leads to cell cycle arrest and cell death. Using a Vav1-Cre based PRMT5 knockout mouse model that we generated, we have found that PRMT5 knockout induces specific dysregulation of RNA splicing, including alternative splicing of the multifunctional epigenetic and DNA repair factor Tip60/KAT5. Aberrant splicing of Tip60 leads to selectively defective acetyl-transferase activity, including histone H4 acetylation, a key event in DNA repair pathway choice. Consistently, we found that ectopic expression of wild-type Tip60 in PRMT5-null cells rescues the homologous recombination phenotype. Thus, we have uncovered a novel interaction between PRMT5-dependent RNA splicing and DNA repair, that functions to regulate genomic stability and cellular homeostasis. Importantly, these findings can be exploited therapeutically as PARP inhibitors have been shown to be effective in HR defective cells, such as BRCA1 or BRCA2 mutant cells. We show here that PRMT5 inhibitors and PARP inhibitors have synergistic effects on leukemia cell lines and mouse models, thus demonstrating the advantages of combining targeted epigenetic and non-epigenetic inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Structural insight into BRCA1-BARD1 complex recruitment to damaged chromatin

The BRCA1-BARD1 complex directs the DNA double-strand break (DSB) repair pathway choice to error-free homologous recombination (HR) during the S-G2 stages. Targeting BRCA1-BARD1 to DSB-proximal sites requires BARD1-mediated nucleosome interaction and histone mark recognition. Here, we report the cryo-EM structure of BARD1 bound to a ubiquitinated nucleosome core particle (NCPUb) at 3.1Å resolution and illustrate how BARD1 simultaneously recognizes the DNA damage-induced mark H2AK15ub and DNA replication-associated mark H4K20me0 on the nucleosome. Invitro and invivo analyses reveal that the BARD1-NCPUb complex is stabilized by BARD1-nucleosome interaction, BARD1-ubiquitin interaction, and BARD1 ARD domain-BARD1 BRCT domain interaction, and abrogating these interactions is detrimental to HR activity. We further identify multiple disease-causing BARD1 mutations that disrupt BARD1-NCPUb interactions and hence impair HR. Together, this study elucidates the mechanism of BRCA1-BARD1 complex recruitment and retention by DSB-flanking nucleosomes and sheds important light on cancer therapeutic avenues.

Sequence modification on demand: search and replace tools for precise gene editing in plants.

Until recently, our ability to generate allelic diversity in plants was limited to introduction of variants from domesticated and wild species by breeding via uncontrolled recombination or the use of chemical and physical mutagens-processes that are lengthy and costly or lack specificity, respectively. Gene editing provides a faster and more precise way to create new variation, although its application in plants has been dominated by the creation of short insertion and deletion mutations leading to loss of gene function, mostly due to the dependence of editing outcomes on DNA repair pathway choices intrinsic to higher eukaryotes. Other types of edits such as point mutations and precise and pre-designed targeted sequence insertions have rarely been implemented, despite providing means to modulate the expression of target genes or to engineer the function and stability of their protein products. Several advancements have been developed in recent years to facilitate custom editing by regulation of repair pathway choices or by taking advantage of alternative types of DNA repair. We have seen the advent of novel gene editing tools that are independent of DNA double-strand break repair, and methods completely independent of host DNA repair processes are being increasingly explored. With the aim to provide a comprehensive review of the state-of-the-art methodology for allele replacement in plants, I discuss the adoption of these improvements for plant genome engineering.

Cornelia\xa0de Lange syndrome-associated mutations cause a DNA damage signalling and repair defect

Cornelia de Lange syndrome is a multisystem developmental disorder typically caused by mutations in the gene encoding the cohesin loader NIPBL. The associated phenotype is generally assumed to be the consequence of aberrant transcriptional regulation. Recently, we identified a missense mutation in BRD4 associated with a Cornelia de Lange-like syndrome that reduces BRD4 binding to acetylated histones. Here we show that, although this mutation reduces BRD4-occupancy at enhancers it does not affect transcription of the pluripotency network in mouse embryonic stem cells. Rather, it delays the cell cycle, increases DNA damage signalling, and perturbs regulation of DNA repair in mutant cells. This uncovers a role for BRD4 in DNA repair pathway choice. Furthermore, we find evidence of a similar increase in DNA damage signalling in cells derived from NIPBL-deficient individuals, suggesting that defective DNA damage signalling and repair is also a feature of typical Cornelia de Lange syndrome.

Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks.

Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays (‘PathSig-dPCR’) for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.

P97/VCP inhibition causes excessive MRE11-dependent DNA end resection promoting cell killing after ionizing radiation.

SummaryThe ATPase p97 is a central component of the ubiquitin-proteasome degradation system. p97 uses its ATPase activity and co-factors to extract ubiquitinated substrates from different cellular locations, including DNA lesions, thereby regulating DNA repair pathway choice. Here, we find that p97 physically and functionally interacts with the MRE11-RAD50-NBS1 (MRN) complex on chromatin and that inactivation of p97 blocks the disassembly of the MRN complex from the sites of DNA damage upon ionizing radiation (IR). The inhibition of p97 function results in excessive 5′-DNA end resection mediated by MRE11 that leads to defective DNA repair and radiosensitivity. In addition, p97 inhibition by the specific small-molecule inhibitor CB-5083 increases tumor cell killing following IR both in vitro and in vivo. Mechanistically, this is mediated via increased MRE11 nuclease accumulation. This suggests that p97 inhibitors might be exploited to improve outcomes for radiotherapy patients.

Early Role of Rif1 in the Repair of P‐element Transposase‐Induced DNA Double Strand Breaks

Rap1 interacting factor (RIF1) is a structurally well-conserved protein in eukaryotes that plays an important role in pathway choice during the repair of DNA double strand breaks (DSBs). In human cells, Rif1 directly interacts with phosphorylated p53 binding protein (53BP1) to inhibit BRCA1 mediated resection of DSBs during G1, and hence promotes repair via non-homologous end joining. In contrast, Saccharomyces cerevisiae Rif1 was shown to promote resection and homology-directed repair of DSBs in both G1 and G2. Given the opposing repair functions of Rif1 in evolutionary distant mammalian and yeast cells, we have explored the DSB repair role of Rif1 in phylogenetic intermediate Drosophila melanogaster. Hatching assays and irradiation sensitivity assays were conducted to determine whether rif1 mutant fly stocks exhibit any DSB repair defect during embryonic development or larval development. No such defect was observed. Next, in order to probe whether Rif1 influences repair pathway choice, as is seen in mammals and yeast, DSB repair assays were conducted. These assays allow for the determination of repair mechanism—end-joining or homology-directed repair—of site-specific DSBs in the male germ-line. rif1 mutants exhibited no defects in the repair of I-SceI endonuclease-induced breaks but, interestingly, a near-complete defect in the repair of P-element transposase-induced breaks. These results suggest that Drosophila RIF1 plays an early role in the repair of P-element transposase-induced breaks and may provide insight into the disparate roles of RIF1 in yeast and mammals.

The Emerging Role of RNA Modifications in DNA Double-Strand Break Repair.

The correct repair of DNA double-strand breaks is essential for maintaining the stability of the genome, thus ensuring the survival and fitness of any living organism. Indeed, the repair of these lesions is a complicated affair, in which several pathways compete for the DNA ends in a complex balance. Thus, the fine-tuning of the DNA double-strand break repair pathway choice relies on the different regulatory layers that respond to environmental cues. Among those different tiers of regulation, RNA modifications have just emerged as a promising field.

Phenotypic Characterization of Sulfolobus islandicus Strains Lacking the B-Family DNA Polymerases PolB2 and PolB3 Individually and in Combination.

Across the three domains of life, B-family DNA polymerases play a variety of roles in both DNA repair and DNA replication processes. We examine the phenotypic consequences of loss of the putative repair polymerases PolB2 and/or PolB3 in the crenarchaeon Sulfolobus islandicus. We detect a modest growth advantage when cells lacking the polymerase are grown in unperturbed conditions. Further, we observe a striking insensitivity of the mutant lines to acute treatment with the oxidizing agent, hydrogen peroxide. In addition, cells lacking PolB3 show enhanced sensitivity to the DNA damaging agent 4-NQO. Our data therefore suggest that these non-essential DNA polymerases may influence DNA repair pathway choice in these hyperthermophilic aerobes.

FANCM regulates repair pathway choice at stalled replication forks

Repair pathway "choice" at stalled mammalian replication forks is an important determinant of genome stability; however, the underlying mechanisms are poorly understood. FANCM encodes a multi-domain scaffolding and motor protein that interacts with several distinct repair protein complexes at stalled forks. Here, we use defined mutations engineered within endogenous Fancm in mouse embryonic stem cells to study how Fancm regulates stalled fork repair. We find that distinct FANCM repair functions are enacted by molecularly separable scaffolding domains. These findings define FANCM as a key mediator of repair pathway choice at stalled replication forks and reveal its molecular mechanism. Notably, mutations that inactivate FANCM ATPase function disable all its repair functions and "trap" FANCM at stalled forks. We find that Brca1 hypomorphic mutants are synthetic lethal with Fancm null or Fancm ATPase-defective mutants. The ATPase function of FANCM may therefore represent a promising "druggable" target for therapy of BRCA1-linked cancer.

N-terminal region of RecQ4 inhibits non-homologous end joining and chromatin association of the Ku heterodimer in Xenopus egg extracts.

RecQ4, a member of the RecQ helicase family, is required for the maintenance of genome integrity. RecQ4 has been shown to promote the following two DNA double-strand break (DSB) repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). However, its molecular function has not been fully elucidated. In the present study, we aimed to investigate the role of RecQ4 in NHEJ using Xenopus egg extracts. The N-terminal 598 amino acid region of Xenopus RecQ4 (N598), which lacks a central helicase domain and a downstream C-terminal region, was added to the extracts and its effect on the joining of DNA ends was analyzed. We found that N598 inhibited the joining of linearized DNA ends in the extracts. In addition, N598 inhibited DSB-induced chromatin binding of Ku70, which is essential for NHEJ, while the DSB-induced chromatin binding of the HR-associated proteins, replication protein A (RPA) and Rad51, increased upon the addition of N598. These results suggest that RecQ4 possibly influences the choice of the DSB repair pathway by influencing the association of the Ku heterodimer with the DNA ends.

Abstract PO-026: DNA damage response and repair characterization in CHK2-deficient cancers

Abstract Double strand breaks (DSBs) are the most lethal DNA lesions as they can result in chromosomal rearrangements or loss of genetic material. To detect and repair DNA, cells have developed the DNA damage response, a cascade of proteins in which the checkpoint kinase 2 (CHK2) is involved. CHK2-deficient cancers, such as breast, prostate or colon, might have a compromised response to ionizing radiation (IR), arresting cell cycle at checkpoints or inducing apoptosis in presence of irreparable damage. To understand how the lack of CHK2 is affecting DNA damage response and repair, the human isogenic HCT116 colorectal cancer cell lines proficient (CHK2 WT) and deficient (CHK2 KO) for CHK2 have been characterized. Cell cycle distribution and γH2AX foci formation were assessed by flow cytometry and immunofluorescence, respectively, to determine the differences in cell cycle checkpoints and DSBs infliction and resolution upon IR. Traffic Light Reporter, complemented by immunofluorescence of RAD51 foci, was employed in both CHK2 WT and KO cell lines to identify the preferred DSB repair pathway. Our results indicate that CHK2-proficient and -deficient cell lines do not exhibit significant differences in cell cycle distribution upon IR. On the other hand, CHK2 KO cells display a significant increase in γH2AX foci per cell compared to the CHK2 WT. Upon 1Gy of IR, cells lacking CHK2 increment the number of foci per cell, indicating that DSBs detection occurs independently of the CHK2 status. Furthermore, the Traffic Light Reporter revealed higher homologous recombination (HR) to non-homologous end-joining (NHEJ) ratio in the CHK2 KO cells as compared to their CHK2 WT counterparts, suggesting that the lack of CHK2 stimulates a shift in the DSBs repair pathway choice. These results contrast with the RAD51 foci formation, which seems to be compromised in the CHK2 KO cells in both treated and untreated conditions as compared to the CHK2-proficient cells. Our results suggest that CHK2 status considerably affects cellular DNA damage response and these differences in execution of DNA repair could be exploited in CHK2-deficient cancers. Citation Format: Carmen Muñoz-Maldonado, Aurélie Quintin, Daniel M. Aebersold, Yitzhak Zimmer, Michaela Medová. DNA damage response and repair characterization in CHK2-deficient cancers [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 PO-026.

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.

Behavior of dicentric chromosomes in budding yeast.

DNA double-strand breaks arise in vivo when a dicentric chromosome (two centromeres on one chromosome) goes through mitosis with the two centromeres attached to opposite spindle pole bodies. Repair of the DSBs generates phenotypic diversity due to the range of monocentric derivative chromosomes that arise. To explore whether DSBs may be differentially repaired as a function of their spatial position in the chromosome, we have examined the structure of monocentric derivative chromosomes from cells containing a suite of dicentric chromosomes in which the distance between the two centromeres ranges from 6.5 kb to 57.7 kb. Two major classes of repair products, homology-based (homologous recombination (HR) and single-strand annealing (SSA)) and end-joining (non-homologous (NHEJ) and micro-homology mediated (MMEJ)) were identified. The distribution of repair products varies as a function of distance between the two centromeres. Genetic dependencies on double strand break repair (Rad52), DNA ligase (Lif1), and S phase checkpoint (Mrc1) are indicative of distinct repair pathway choices for DNA breaks in the pericentromeric chromatin versus the arms.