Repair DNA Double-strand Breaks Research Articles

Regulation of DNA damage response by trimeric G-proteins

Upon sensing DNA double-strand breaks (DSBs), eukaryotic cells either die or repair DSBs via one of the two competing pathways, i.e., non-homologous end-joining (NHEJ) or homologous recombination (HR). We show that cell fate after DSBs hinges on GIV/Girdin, a guanine nucleotide-exchange modulator of heterotrimeric Giα•βγ protein. GIV suppresses HR by binding and sequestering BRCA1, akey coordinator of multiple steps within the HR pathway, away from DSBs; itdoes so using a C-terminal motif that binds BRCA1's BRCT-modules via both phospho-dependent and -independent mechanisms. Using another non-overlapping C-terminal motif GIV binds and activates Gi and enhances the "free" Gβγ→PI-3-kinase→Akt pathway, which promotes survival and is known to suppress HR, favor NHEJ. Absence of GIV, or loss of either of its C-terminal motifs enhanced cell death upon genotoxic stress. Because GIV selectively binds other BRCT-containing proteins suggests that G-proteins may fine-tune sensing, repair, and survival after diverse types of DNA damage.

An Expansion of the Endoplasmic Reticulum that Halts Autophagy is Permissive to Genome Instability.

The links between autophagy and genome stability, and whether they are important for lifespan and health, are not fully understood. We undertook a study to explore this notion at the molecular level using Saccharomyces cerevisiae. On the one hand, we triggered autophagy using rapamycin, to which we exposed mutants defective in preserving genome integrity, then assessed their viability, their ability to induce autophagy and the link between these two parameters. On the other hand, we searched for molecules derived from plant extracts known to have powerful benefits on human health to try to rescue the negative effects rapamycin had against some of these mutants. We uncover that autophagy execution is lethal for mutants unable to repair DNA double strand breaks, while the extract from Silybum marianum seeds induces an expansion of the endoplasmic reticulum (ER) that blocks autophagy and protects them. Our data uncover a connection between genome integrity and homeostasis of the ER whereby ER stress-like scenarios render cells tolerant to sub-optimal genome integrity conditions.

Structural basis of DNA polymerase θ mediated DNA end joining.

DNA polymerase θ (Pol θ) plays an essential role in the microhomology-mediated end joining (MMEJ) pathway for repairing DNA double-strand breaks. However, the mechanisms by which Pol θ recognizes microhomologous DNA ends and performs low-fidelity DNA synthesis remain unclear. Here, we present cryo-electron microscope structures of the polymerase domain of Lates calcarifer Pol θ with long and short duplex DNA at up to 2.4 Å resolution. Interestingly, Pol θ binds to long and short DNA substrates similarly, with extensive interactions around the active site. Moreover, Pol θ shares a similar active site as high-fidelity A-family polymerases with its finger domain well-closed but differs in having hydrophilic residues surrounding the nascent base pair. Computational simulations and mutagenesis studies suggest that the unique insertion loops of Pol θ help to stabilize short DNA binding and assemble the active site for MMEJ repair. Taken together, our results illustrate the structural basis of Pol θ-mediated MMEJ.

Selective utilization of non-homologous end-joining and homologous recombination for DNA repair during meiotic maturation in mouse oocytes.

DNA double-strand breaks (DSBs) are highly toxic lesions that can cause genomic instability and can be repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways. Despite extensive studies about DSB repair pathways, the roles of each pathway during meiotic maturation in oocytes are not well understood. Here we show that oocytes selectively utilize NHEJ and HR to repair DSBs during meiotic maturation. Inhibition of NHEJ impaired the meiotic maturation of oocytes with DNA damage by activating the spindle assembly checkpoint (SAC) with a concomitant increase in metaphase I (MI) arrest and DNA damage levels. In contrast, oocytes with DNA damage bypassed SAC-mediated MI arrest despite the presence of fragmented DNA when HR was inhibited. Notably, this bypass of SAC arrest by HR inhibition was associated with a loss of centromere integrity and subsequent impairment of chromosome architecture. Our results demonstrate that, while NHEJ is critical for the meiotic maturation of oocytes with DNA damage, HR is essential to maintain centromere integrity against DNA damage during meiotic maturation, revealing distinct roles of NHEJ and HR during meiotic maturation in mouse oocytes.

FBXW7 alleviates hyperglycemia-induced endothelial oxidative stress injury via ROS and PARP inhibition

Diabetic retinopathy (DR) and other diabetic vascular complications are the leading cause of death and disability in patients with suboptimum glycemic control. In the pathogenesis of diabetic vascular diseases, hyperglycemia-induced oxidative stress, DNA damage, and poly-ADP-ribose-polymerase (PARP) hyperactivation play important roles in endothelial cell impairment. Adipose differentiation-related protein FBXW7 was reported to regulate PGC-1α stability and mitochondrial homeostasis. Here, we investigated the role and mechanism of FBXW7 in repairing endothelial oxidative stress injuries under hyperglycemic conditions. FBXW7 promoted the hampered activity of homologous recombination and non-homologues end joining pathway for repairing DNA double-strand breaks damage, an initiating factor for PARP hyperactivation and diabetic vascular complications. The abundant mobilization of DNA damage repair mediated by FBXW7 suppressed PARP activation, leading to downregulation of PARP expression and activity in both human endothelial cells and diabetic rat retinas. This provided a new method for PARP inhibition, superior to PARP inhibitors for treating diabetic vascular complication. Furthermore, FBXW7 rescued downregulated NAD+ levels and ameliorated mitochondrial dysfunction, thereby reducing superoxide production under hyperglycemic conditions. These effects reversed oxidative injury and vascular leakage in diabetic rat retina, providing a potential future treatment strategy.

Assessing the Effects of Inherited Mutations on PALB2 Structure and Function

The proper functioning of the protein PALB2 is vital to preventing tumor formation within breast tissues in individuals. Upon the detection of DNA damage, PALB2 and BRCA1 bind to each other along with BRCA2 to form a DNA repair complex. This complex then repairs DNA double-strand breaks in order to prevent the accumulation of DNA damage that leads to breast cancer. While both BRCA1 and BRCA2 have been extensively studied, a lot of information about the structure and function of PALB2 remains unknown. It is thought that BRCA1 and PALB2 bind via PALB2's coiled-coil domain; however, how variants of unknown significance (VUS) affect this binding interaction is largely unknown. Further, while some of these VUS have been studied in vivo, cheaper and easier in vitro methods to measure their effect on binding affinity have yet to be formulated. Thus, we hypothesized that isothermal titration calorimetry (ITC) could be used as an in vitro testing method for assessing the effects of VUS within the coiled-coil domain of PALB2 on the binding event between PALB2 and BRCA1. Further, we hypothesized that a decrease in binding between the two proteins as measured by ITC would correlate with a decrease in DNA repair as measured in vivo. We tested the efficacy of this method by creating seven mutations within the coiled-coil domain of PALB2 and measuring the binding event of PALB2 to BRCA1 via ITC. Our results strongly suggest that the binding event is enthalpic in nature and can be adequately measured using ITC as evidenced by the correlation between our in vitro data and previous in vivo data.

Increased Gene Targeting in Hyper-Recombinogenic LymphoBlastoid Cell Lines Leaves Unchanged DSB Processing by Homologous Recombination.

In the cells of higher eukaryotes, sophisticated mechanisms have evolved to repair DNA double-strand breaks (DSBs). Classical nonhomologous end joining (c-NHEJ), homologous recombination (HR), alternative end joining (alt-EJ) and single-strand annealing (SSA) exploit distinct principles to repair DSBs throughout the cell cycle, resulting in repair outcomes of different fidelity. In addition to their functions in DSB repair, the same repair pathways determine how cells integrate foreign DNA or rearrange their genetic information. As a consequence, random integration of DNA fragments is dominant in somatic cells of higher eukaryotes and suppresses integration events at homologous genomic locations, leading to very low gene-targeting efficiencies. However, this response is not universal, and embryonic stem cells display increased targeting efficiency. Additionally, lymphoblastic chicken and human cell lines DT40 and NALM6 show up to a 1000-fold increased gene-targeting efficiency that is successfully harnessed to generate knockouts for a large number of genes. We inquired whether the increased gene-targeting efficiency of DT40 and NALM6 cells is linked to increased rates of HR-mediated DSB repair after exposure to ionizing radiation (IR). We analyzed IR-induced γ-H2AX foci as a marker for the total number of DSBs induced in a cell and RAD51 foci as a marker for the fraction of those DSBs undergoing repair by HR. We also evaluated RPA accretion on chromatin as evidence for ongoing DNA end resection, an important initial step for all pathways of DSB repair except c-NHEJ. We finally employed the DR-GFP reporter assay to evaluate DSB repair by HR in DT40 cells. Collectively, the results obtained, unexpectedly show that DT40 and NALM6 cells utilized HR for DSB repair at levels very similar to those of other somatic cells. These observations uncouple gene-targeting efficiency from HR contribution to DSB repair and suggest the function of additional mechanisms increasing gene-targeting efficiency. Indeed, our results show that analysis of the contribution of HR to DSB repair may not be used as a proxy for gene-targeting efficiency.

SETD2 regulates gene transcription patterns and is associated with radiosensitivity in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) has high morbidity and mortality worldwide, and its prognosis remains unsatisfactory. Identification of epigenetic biomarkers associated with radiosensitivity is beneficial for precision medicine in LUAD patients. SETD2 is important in repairing DNA double-strand breaks and maintaining chromatin integrity. Our studies established a comprehensive analysis pipeline, which identified SETD2 as a radiosensitivity signature. Multi-omics analysis revealed enhanced chromatin accessibility and gene transcription by SETD2. In both LUAD bulk RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq), we found that SETD2-associated positive transcription patterns were associated with DNA damage responses. SETD2 knockdown significantly upregulated tumor cell apoptosis, attenuated proliferation and migration of LUAD tumor cells, and enhanced radiosensitivity in vitro. Moreover, SETD2 was a favorably prognostic factor whose effects were antagonized by the m6A-related genes RBM15 and YTHDF3 in LUAD. In brief, SETD2 was a promising epigenetic biomarker in LUAD patients.

The regulation of DNA end resection by chromatin response to DNA double strand breaks.

DNA double-strand breaks (DSBs) constantly arise upon exposure to genotoxic agents and during physiological processes. The timely repair of DSBs is important for not only the completion of the cellular functions involving DSBs as intermediates, but also the maintenance of genome stability. There are two major pathways dedicated to DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). The decision of deploying HR or NHEJ to repair DSBs largely depends on the structures of broken DNA ends. DNA ends resected to generate extensive single-strand DNA (ssDNA) overhangs are repaired by HR, while those remaining blunt or minimally processed can be repaired by NHEJ. As the generation and repair of DSB occurs within the context of chromatin, the resection of broken DNA ends is also profoundly affected by the state of chromatin flanking DSBs. Here we review how DNA end resection can be regulated by histone modifications, chromatin remodeling, and the presence of ssDNA structure through altering the accessibility to chromatin and the activity of pro- and anti-resection proteins.

Experience with olaparib in a patient with luminal HER2-positive metastatic breast cancer

Hereditary breast cancer (BC) accounts for about 5-10% of cases. BRCA-associated tumors have been identified as a separate group of malignant neoplasms with distinctive clinical manifestations and specific treatment features. Understanding of biological mechanisms leading to cancer in BRCA1/2 mutation carriers and discovery of potential molecular targets, such as poly (ADP-ribose) polymerase (PARP), involved in base excision repair mechanisms, led to the development of a new class of targeted drugs belonging to the PARP inhibitors group. PARP inhibition leads to the preservation of single-stranded DNA breaks, the arrest of the replication fork, and the realization of the “synthetic lethality” phenomenon due to the inability to repair double-stranded DNA breaks by homologous recombination in cells with mutations in the BRCA1/2 genes. Two randomized trials OlympiAD and EMBRACA evaluated and proved the effectiveness of PARP inhibitors in patients with metastatic BRCA-mutated HER2-negative breast cancer in comparison with standard chemotherapy. At the same time, data on the potential use of PARP inhibitors for the treatment of BRCA-mutated HER2-positive breast cancer patients are extremely limited. This article presents a clinical example of the use of olaparib in a patient with BRCA-mutated HER2-positive metastatic breast cancer.

Phase 1 results of a phase 1/2 trial of CYT-0851, a first-in-class inhibitor of RAD51-mediated homologous recombination, in patients with advanced solid and hematologic cancers.

3084 Background: Homologous recombination (HR) is an essential, high-fidelity mechanism to repair DNA double strand breaks (DSBs) in cancer cells. CYT-0851 inhibits HR leading to an accumulation of unrepaired DSBs and tumor cell death. We are reporting the completed Phase 1 dose-escalation results and RP2D selection to support ongoing development of CYT-0851. Methods: Patients (pts) with advanced hematologic and solid tumors were treated with continuous 28-day cycles of increasing doses of CYT-0851 following a 3+3 design. The primary objective was determining the maximum tolerated dose. Secondary objectives included safety, pharmacokinetics, and anti-tumor activity. Results: As of a 15 Nov 2021 data cutoff (DCO), 73 pts with advanced cancers (NHL n = 18; Sarcoma n = 16; Pancreas n = 11; Breast n = 8; HNSCC n = 6; Ovarian n = 4; SCLC n = 4; Other n = 4; Myeloma n = 2) were treated in 12 cohorts (total daily doses: 30 mg to 1200 mg). One pt experienced a dose-limiting toxicity (DLT) of reversible metabolic acidosis at 1200 mg. Two of 3 pts at 800 mg experienced reversible DLT-like events in Cycle 2 of Gr3 dry skin and Gr3 myalgia and polyarthritis, respectively. Three of 10 pts treated at 600 mg experienced DLT-like events in Cycle 1: 1 pt experienced an SAE of Gr3 anorexia with Gr3 stomatitis, vomiting, and dehydration and 2 pts had Gr3 fatigue. No DLTs occurred at 400 mg daily which was selected as the RP2D. 42 pts (57.5%) experienced a CYT-0851-related adverse event (AE), including 12 (16.4%) with a Gr3/4 AE. There were no treatment-related deaths. AE leading to CYT-0851 withdrawal were reported in 2 pts (2.7%) treated with 600 and 800 mg. The most common CYT-0851-related AEs were primarily Gr1/2 and included fatigue (20.5%), hyperuricemia (11%), nausea (11%), alopecia (9.6%), constipation (8.2%) and headache (8.2%). CYT-0851 exposure was approximately dose-proportional across the evaluated doses with an effective half-life of ̃3 days. Exposure at 400 mg daily was consistent with efficacy in preclinical models. 46 pts were evaluable for response at the DCO. 12 pts with NHL were evaluable by Lugano and included 1 CR in FL and 1 PR in DLBCL treated for 393+ and 244 days respectively. 34 pts with solid tumors were evaluable by RECIST v1.1 with 1 PR in a pt with myxofibrosarcoma treated for 313 days and 16 pts with stable disease. Fifteen pts were treated for 100+ days and 5 for 180+ days. Conclusions: CYT-0851 has demonstrated promising and broad clinical activity in a Ph 1 population of pts with advanced cancers. The safety profile is favorable as characterized by events that were infrequent, primarily Gr1/2, and reversible. Six expansion cohorts (DLBCL, Follicular Lymphoma, Myeloma, Pancreatic, Ovarian and Sarcoma) are enrolling to characterize activity at the RP2D. Ph 1 evaluation of CYT-0851 in combination with 3 chemotherapy backbones is also ongoing. Clinical trial information: NCT03997968.

The unified approach of radiation action on biological matter: The induction of oncogenic transformation by heavy charged particles in C3H10T1/2 cells

The induction of oncogenic transformation to C3H10T1/2 cells by different types of ionizing radiation has been widely studied in various radiological laboratories. Based on the information available in literature, a database is structured to include radiological parameters, that manifest oncogenic effects as well as cellular inactivation of C3H10T1/2 cells, resulted from exposure to different types of heavy charged ions including neutrons. We find that oncogenic transformation effective cross-section is best correlated with mean free path for linear primary ionization. A simple radiobiological model is proposed merely to quantize cross-sections against mean free path. The model reveals saturations of; cellular inactivation cross-section of about 75 mm2, and oncogenic transformation cross-section of about 3.98 x 10-2 mm2, both started at mean free path of 1.8 nm (inflection points) and lower values. Since the interspacing distance between the DNA strands is about 1.8 nm, the model explains the crucial roles of DNA lesions (caused by heavy charged particles) to play as the starting point leading to cell death or oncogenic transformation. The effective cross -sections in the sloping regions are primarily due to repairable DNA single strand breaks while saturation regions are essentially due to unrepaired or incorrectly repaired DNA double strand breaks.

In vitro dose effect relationships of actinium-225- and lutetium-177-labeled PSMA-I&T

PurposeTargeting the prostate-specific membrane antigen (PSMA) using lutetium-177-labeled PSMA-specific tracers has become a very promising novel therapy option for prostate cancer (PCa). The efficacy of this therapy might be further improved by replacing the β-emitting lutetium-177 with the α-emitting actinium-225. Actinium-225 is thought to have a higher therapeutic efficacy due to the high linear energy transfer (LET) of the emitted α-particles, which can increase the amount and complexity of the therapy induced DNA double strand breaks (DSBs). Here we evaluated the relative biological effectiveness of [225Ac]Ac-PSMA-I&T and [177Lu]Lu-PSMA-I&T by assessing in vitro binding characteristics, dosimetry, and therapeutic efficacy.Methods and resultsThe PSMA-expressing PCa cell line PC3-PIP was used for all in vitro assays. First, binding and displacement assays were performed, which revealed similar binding characteristics between [225Ac]Ac-PSMA-I&T and [177Lu]Lu-PSMA-I&T. Next, the assessment of the number of 53BP1 foci, a marker for the number of DNA double strand breaks (DSBs), showed that cells treated with [225Ac]Ac-PSMA-I&T had slower DSB repair kinetics compared to cells treated with [177Lu]Lu-PSMA-I&T. Additionally, clonogenic survival assays showed that specific targeting with [225Ac]Ac-PSMA-I&T and [177Lu]Lu-PSMA-I&T caused a dose-dependent decrease in survival. Lastly, after dosimetric assessment, the relative biological effectiveness (RBE) of [225Ac]Ac-PSMA-I&T was found to be 4.2 times higher compared to [177Lu]Lu-PSMA-I&T.ConclusionWe found that labeling of PSMA-I&T with lutetium-177 or actinium-225 resulted in similar in vitro binding characteristics, indicating that the distinct biological effects observed in this study are not caused by a difference in uptake of the two tracers. The slower repair kinetics of [225Ac]Ac-PSMA-I&T compared to [177Lu]Lu-PSMA-I&T correlates to the assumption that irradiation with actinium-225 causes more complex, more difficult to repair DSBs compared to lutetium-177 irradiation. Furthermore, the higher RBE of [225Ac]Ac-PSMA-I&T compared to [177Lu]Lu-PSMA-I&T underlines the therapeutic potential for the treatment of PCa.

Differential Expression of Nuclear Body Component, Zc3h8, Disrupts DNA Double Strand Break Repair

The processes of DNA damage recognition and repair are paramount to maintaining genome integrity in the cell. Failure to do so results in genetic instability and the accumulation of mutations. Double strand breaks have the greatest potential to introduce mutations because rearrangements, non‐homologous end joining, deletions, or insertions may result. Unrepaired or incorrectly repaired DNA double strand breaks can lead to cell death, apoptosis, or disease such as cancer. We previously demonstrated that the C3H1 zinc finger‐containing protein Zc3h8 (also called Fliz1) promotes oncogenic phenotypes when overexpressed in cells, while oncogenic phenotypes are suppressed when expression is reduced. In mice, Zc3h8 expression affected tumorigenesis and tumor size in vivo. Higher expression of Zc3h8 led to more frequent tumors and larger tumors in mice. In humans Zc3h8 is overexpressed in 2‐6% of cancers and is a statistically significant prognostic indicator for poor rates of survival. The Zc3h8 protein has been localized to nuclear bodies including promyelocytic leukemia (PML) and Cajal Bodies where key cellular processes including transcription, translation, post‐translational modifications, and RNA processing are regulated. PML Bodies, in particular, have a role in DNA damage response and repair since P53 is located in PML Bodies along with DAXX, ATRX, and CK2. Since Zc3h8 co‐localized with DNA damage response and repair proteins in PML bodies and promotes oncogenic properties in cells, we investigated if Zc3h8 expression alters how cells respond to DNA damage such as double strand breaks and how this damage is mitigated in cells. Differential expression of Zc3h8 affected mouse mammary tumor cell’s ability to repair double strand breaks caused by the topoisomerase II inhibitor etoposide as indicated by the presence of γH2AX, a marker for unrepaired double strand breaks. Using a Comet assay, we found extensive DNA fragmentation in cells with elevated Zc3h8 expression despite high rates of proliferation and migration. Furthermore, Zc3h8 increases cellular tolerance for DNA damage and alters apoptosis. These phenotypes promote further mutation and may indicate a molecular cause for highly aggressive and progressive cancers with elevated Zc3h8 expression. These conclusions suggest a DNA damage repair and response pathway that is altered by Zc3h8 expression levels can affect multiple aspects of cellular behavior and promote oncogenic phenotypes.

Failure of DNA double-strand break repair by tau mediates Alzheimer\u2019s disease pathology in vitro

DNA double-strand break (DSB) is the most severe form of DNA damage and accumulates with age, in which cytoskeletal proteins are polymerized to repair DSB in dividing cells. Since tau is a microtubule-associated protein, we investigate whether DSB is involved in tau pathologies in Alzheimer’s disease (AD). First, immunohistochemistry reveals the frequent coexistence of DSB and phosphorylated tau in the cortex of AD patients. In vitro studies using primary mouse cortical neurons show that non-p-tau accumulates perinuclearly together with the tubulin after DSB induction with etoposide, followed by the accumulation of phosphorylated tau. Moreover, the knockdown of endogenous tau exacerbates DSB in neurons, suggesting the protective role of tau on DNA repair. Interestingly, synergistic exposure of neurons to microtubule disassembly and the DSB strikingly augments aberrant p-tau aggregation and apoptosis. These data suggest that DSB plays a pivotal role in AD-tau pathology and that the failure of DSB repair leads to tauopathy.

Building Biological Relevance Into Integrative Modelling of Macromolecular Assemblies.

Recent advances in structural biophysics and integrative modelling methods now allow us to decipher the structures of large macromolecular assemblies. Understanding the dynamics and mechanisms involved in their biological function requires rigorous integration of all available data. We have developed a complete modelling pipeline that includes analyses to extract biologically significant information by consistently combining automated and interactive human-guided steps. We illustrate this idea with two examples. First, we describe the ryanodine receptor, an ion channel that controls ion flux across the cell membrane through transitions between open and closed states. The conformational changes associated with the transitions are small compared to the considerable system size of the receptor; it is challenging to consistently track these states with the available cryo-EM structures. The second example involves homologous recombination, in which long filaments of a recombinase protein and DNA catalyse the exchange of homologous DNA strands to reliably repair DNA double-strand breaks. The nucleoprotein filament reaction intermediates in this process are short-lived and heterogeneous, making their structures particularly elusive. The pipeline we describe, which incorporates experimental and theoretical knowledge combined with state-of-the-art interactive and immersive modelling tools, can help overcome these challenges. In both examples, we point to new insights into biological processes that arise from such interdisciplinary approaches.

Genome-wide investigation of maize RAD51 binding affinity through phage display

BackgroundRAD51 proteins, which are conserved in all eukaryotes, repair DNA double-strand breaks. This is critical to homologous chromosome pairing and recombination enabling successful reproduction. Work in Arabidopsis suggests that RAD51 also plays a role in plant defense; the Arabidopsis rad51 mutant is more susceptible to Pseudomonas syringae. However, the defense functions of RAD51 and the proteins interacting with RAD51 have not been thoroughly investigated in maize. Uncovering ligands of RAD51 would help to understand meiotic recombination and possibly the role of RAD51 in defense. This study used phage display, a tool for discovery of protein-protein interactions, to search for proteins interacting with maize RAD51A1.ResultsMaize RAD51A1 was screened against a random phage library. Eleven short peptide sequences were recovered from 15 phages which bound ZmRAD51A1 in vitro; three sequences were found in multiple successfully binding phages. Nine of these phage interactions were verified in vitro through ELISA and/or dot blotting.BLAST searches did not reveal any maize proteins which contained the exact sequence of any of the selected phage peptides, although one of the selected phages had a strong alignment (E-value = 0.079) to a binding domain of maize BRCA2. Therefore, we designed 32 additional short peptides using amino acid sequences found in the predicted maize proteome. These peptides were not contained within phages. Of these synthesized peptides, 14 bound to ZmRAD51A1 in a dot blot experiment. These 14 sequences are found in known maize proteins including transcription factors putatively involved in defense.ConclusionsThese results reveal several peptides which bind ZmRAD51A1 and support a potential role for ZmRAD51A1 in transcriptional regulation and plant defense. This study also demonstrates the applicability of phage display to basic science questions, such as the search for binding partners of a known protein, and raises the possibility of an iterated approach to test peptide sequences that closely but imperfectly align with the selected phages.

ZMYM2 restricts 53BP1 at DNA double-strand breaks to favor BRCA1 loading and homologous recombination.

An inability to repair DNA double-strand breaks (DSBs) threatens genome integrity and can contribute to human diseases, including cancer. Mammalian cells repair DSBs mainly through homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice between these pathways is regulated by the interplay between 53BP1 and BRCA1, whereby BRCA1 excludes 53BP1 to promote HR and 53BP1 limits BRCA1 to facilitate NHEJ. Here, we identify the zinc-finger proteins (ZnF), ZMYM2 and ZMYM3, as antagonizers of 53BP1 recruitment that facilitate HR protein recruitment and function at DNA breaks. Mechanistically, we show that ZMYM2 recruitment to DSBs and suppression of break-associated 53BP1 requires the SUMO E3 ligase PIAS4, as well as SUMO binding by ZMYM2. Cells deficient for ZMYM2/3 display genome instability, PARP inhibitor and ionizing radiation sensitivity and reduced HR repair. Importantly, depletion of 53BP1 in ZMYM2/3-deficient cells rescues BRCA1 recruitment to and HR repair of DSBs, suggesting that ZMYM2 and ZMYM3 primarily function to restrict 53BP1 engagement at breaks to favor BRCA1 loading that functions to channel breaks to HR repair. Identification of DNA repair functions for these poorly characterized ZnF proteins may shed light on their unknown contributions to human diseases, where they have been reported to be highly dysregulated, including in several cancers.

Meiotic Genes and DNA Double Strand Break Repair in Cancer.

Tumor cells show widespread genetic alterations that change the expression of genes driving tumor progression, including genes that maintain genomic integrity. In recent years, it has become clear that tumors frequently reactivate genes whose expression is typically restricted to germ cells. As germ cells have specialized pathways to facilitate the exchange of genetic information between homologous chromosomes, their aberrant regulation influences how cancer cells repair DNA double strand breaks (DSB). This drives genomic instability and affects the response of tumor cells to anticancer therapies. Since meiotic genes are usually transcriptionally repressed in somatic cells of healthy tissues, targeting aberrantly expressed meiotic genes may provide a unique opportunity to specifically kill cancer cells whilst sparing the non-transformed somatic cells. In this review, we highlight meiotic genes that have been reported to affect DSB repair in cancers derived from somatic cells. A better understanding of their mechanistic role in the context of homology-directed DNA repair in somatic cancers may provide useful insights to find novel vulnerabilities that can be targeted.

RNA Interference Induces BRCA1 Gene Methylation and Increases the Radiosensitivity of Breast Cancer Cells.

Purpose: To investigate the relationship between breast cancer susceptibility gene-1 (BRCA1) gene methylation and the radiosensitivity of breast cancer. Materials and Methods: The authors studied three breast cancer cell lines: MDA-MB-435, MDA-MB-231, and MCF-7 cells. They constructed five short hairpin RNAs (shRNAs) and five small interfering RNAs to target selected promoter loci and initiate sequence-specific methylation in breast cancer cells. Pyrosequencing was used to analyze the state of DNA methylation. Quantitative real-time polymerase chain reaction was used to detect BRCA1 mRNA expression and RNA-directed DNA methylation (RdDM)-related gene expression. Western blotting was performed to analyze BRCA1 protein expression. Colony formation assays and γ-histone H2A foci formation assays were conducted to assess the surviving fraction (SF) and double-strand break (DSB) repair ability of cells after irradiation. Results: The authors constructed five strains of lentivirus vectors and five plasmid vectors targeting BRCA1 promoter region. In MDA-MB-435 cells, lentivirus-mediated RNA interference targeting Site 1 of BRCA1 increased the methylation levels of BRCA1 and reduced BRCA1 mRNA and protein expression. The SF and the ability to repair DNA DSBs were reduced in the combined LV-BRCA1RNAi-Site 1 infection and irradiation group. Conclusions: The authors' findings suggest that the shRNA suppressed the expression levels of the BRCA1 gene and reduced the SF and DNA repair ability of cells after irradiation through RdDM. In summary, the radiosensitivity of breast cancer cells may correlate with BRCA1 methylation. Advances in Knowledge: The authors first utilized a lentivirus-based shRNA-mediated specific-sequence DNA methylation of the BRCA1 gene mediated by RdDM.