Laser Microirradiation Research Articles

Assessment of epigenetic mechanisms and DNA double-strand break repair using laser micro-irradiation technique developed for hematological cells.

BackgroundCertain tumors rely heavily on their DNA repair capability to survive the DNA damage induced by chemotherapeutic agents. Therefore, it is important to monitor the dynamics of DNA repair in patient samples during the course of their treatment, in order to determine whether a particular drug regimen perturbs the DNA repair networks in cancer cells and provides therapeutic benefits. Quantitative measurement of proteins and/or their posttranslational modification(s) at DNA double strand breaks (DSBs) induced by laser microirradiation provides an applicable diagnostic approach to examine DNA repair and its dynamics. However, its use is restricted to adherent cell lines and not employed in suspension tumor cells that include the many hematological malignancies.MethodsHere, we report the development of an assay to laser micro-irradiate and quantitatively measure DNA repair transactions at DSB sites in normal mononuclear cells and a variety of suspension leukemia and lymphoma cells including primary patient samples.FindingsWe show that global changes in the H3K27me3-ac switch modulated by inhibitors of Class I HDACs, EZH2 methyltransferase and (or) H3K27me3 demethylases do not reflect the dynamic changes in H3K27me3 that occur at double-strand break sites during DNA repair.InterpretationResults from our mechanistic studies and proof-of-principle data with patient samples together show the effectiveness of using the modified micro-laser-based assay to examine DNA repair directly in suspension cancer cells, and has important clinical implications by serving as a valuable tool to assess drug efficacies in hematological cancer cells that grow in suspension.

Pellino1 specifically binds to phospho-Thr18 of p53 and is recruited to sites of DNA damage

Pellino1 is an E3 ubiquitin ligase that plays a key role in positive regulation of innate immunity signaling, specifically required for the production of interferon when induced by viral double-stranded RNA. We report the identification of the tumor suppressor protein, p53, as a binding partner of Pellino1. Their interaction has a Kd of 42 ± 2 μM and requires phosphorylation of Thr18 within p53 and association with the forkhead-associated (FHA) domain of Pellino1. We employed laser micro-irradiation and live cell microscopy to show that Pellino1 is recruited to newly occurring DNA damage sites, via its FHA domain. Mutation of a hitherto unidentified nuclear localization signal within the N-terminus of Pellino1 led to its exclusion from the nucleus. This study provides evidence that Pellino1 translocates to damaged DNA in the nucleus and has a functional role in p53 signaling and the DNA damage response.

High-throughput Evaluation of Protein Migration and Localization after Laser Micro-Irradiation

DNA- and histone-related research frequently comprises the quantitative analysis of protein modifications, such as histone phosphorylation. Analysis of accumulation and disappearance of protein foci are used to monitor DNA damage and repair kinetics. If the protein of interest doesn’t accumulate in foci, laser micro-irradiation of single nuclei provides an alternative method to monitor DNA repair proteins and histone dynamics at the DNA damage site. We have developed an automated evaluation tool for standardized, high-throughput analysis of micro-irradiated cells featuring single cell background subtraction and detection across multiple fluorescence channels, allowing for robust statistics.

Assessment of DNA Double-Strand Break Repair at Laser-Induced Damage Sites in Hematological Cancer Cells

Certain tumors rely heavily on their DNA repair capability to survive the DNA damage induced by chemotherapeutic agents. Therefore, it is important to monitor the dynamics of DNA repair in patient samples during the course of their treatment, in order to determine whether a particular drug regimen perturbs the DNA repair networks in cancer cells and provides therapeutic benefits. Quantitative measurement of proteins and/or their post-translational modification(s) at DNA double strand breaks (DSBs) induced by laser micro-irradiation provides an applicable diagnostic approach to examine DNA repair and its dynamics. However, its use is restricted to adherent cell lines and not employed in suspension tumor cells that include the many hematological malignancies. Here, we now report a method to laser micro-irradiate and quantitatively measure DNA repair transactions at DSB sites in a variety of suspension leukemia and lymphoma cells including primary patient samples. We have also used our refined micro-laser method to examine the effects of small molecule inhibitors of epigenetic histone-modifying enzymes in modulating the levels of histone H3 lysine-27 trimethylation (H3K27me3) at DSB sites during DNA repair in a subset of DNA repair 'addicted' diffuse large B-cell lymphoma (DLBCL) cells. Results from our mechanistic studies and proof-of-principle data with patient samples together show the effectiveness of using the modified micro-laser-based assay to examine DNA repair directly in suspension cancer cells, and has important clinical implications by serving as a valuable tool to assess drug efficacies in cancer cells that grow in suspension. Funding: This work was supported by National Institute of Health grants (1R011CA188520 and R01CA1885200S1) as well as Huntsman Cancer Institute and Radiation Oncology development funds to SB. Declaration of Interest: The authors have no competing financial interests. Ethical Approval: The patient sample culture was carried out in accordance with the University of Utah IRB guidelines. The samples are de-identified samples and hence we do not have access to the patient history.

Processive DNA synthesis is associated with localized decompaction of constitutive heterochromatin at the sites of DNA replication and repair

ABSTRACTConstitutive heterochromatin is considered as a functionally inert genome compartment, important for its architecture and stability. How such stable structure is maintained is not well understood. Here, we apply four different visualization schemes to label it and investigate its dynamics during DNA replication and repair. We show that replisomes assemble over the heterochromatin in a temporally ordered manner. Furthermore, heterochromatin undergoes transient decompaction locally at the active sites of DNA synthesis. Using selective laser microirradiation conditions that lead to damage repaired via processive DNA synthesis, we measured similarly local decompaction of heterochromatin. In both cases, we could not observe large-scale movement of heterochromatin to the domain surface. Instead, the processive DNA synthesis machinery assembled at the replication/repair sites. Altogether, our data are compatible with a progression of DNA replication/repair along the chromatin in a dynamic mode with localized and transient decompaction that does not globally remodels the whole heterochromatin compartment.

Domain analysis of PNKP–XRCC1 interactions: Influence of genetic variants of XRCC1

Polynucleotide kinase/phosphatase (PNKP) and X-ray repair cross-complementing 1 (XRCC1) are key proteins in the single-strand DNA break repair pathway. Phosphorylated XRCC1 stimulates PNKP by binding to its forkhead-associated (FHA) domain, whereas nonphosphorylated XRCC1 stimulates PNKP by interacting with the PNKP catalytic domain. Here, we have further studied the interactions between these two proteins, including two variants of XRCC1 (R194W and R280H) arising from single-nucleotide polymorphisms (SNPs) that have been associated with elevated cancer risk in some reports. We observed that interaction of the PNKP FHA domain with phosphorylated XRCC1 extends beyond the immediate, well-characterized phosphorylated region of XRCC1 (residues 515-526). We also found that an XRCC1 fragment, comprising residues 166-436, binds tightly to PNKP and DNA and efficiently activates PNKP's kinase activity. However, interaction of either of the SNP-derived variants of this fragment with PNKP was considerably weaker, and their stimulation of PNKP was severely reduced, although they still could bind DNA effectively. Laser microirradiation revealed reduced recruitment of PNKP to damaged DNA in cells expressing either XRCC1 variant compared with PNKP recruitment in cells expressing WT XRCC1 even though WT and variant XRCC1s were equally efficient at localizing to the damaged DNA. These findings suggest that the elevated risk of cancer associated with these XRCC1 SNPs reported in some studies may be due in part to the reduced ability of these XRCC1 variants to recruit PNKP to damaged DNA.

RAD51 regulates REV1 recruitment to DNA double-strand break sites

REV1 is one of the major Y-family DNA polymerases. It not only functions as a scaffold protein to mediate other specialized DNA polymerases to sites of lesions, but also inserts deoxycytidine across the lesion strand during translesion DNA synthesis (TLS). Meanwhile, REV1 has been reported to be involved in homologous recombination (HR) repair. Here we further explore the roles of REV1-interacting proteins RAD51 and RAD51C in REV1-mediated DNA double-strand break (DSB) repair. We found that RAD51 but not RAD51C regulates REV1 recruitment to DSB sites via pulsed laser microirradiation. Interestingly, immunofluorescence staining exhibits that REV1 also regulates RAD51 focus formation in response to CPT treatment. These results suggest that REV1 and RAD51 might be mutually dependent on each other in the REV1-related HR pathway.

Poly(ADP-ribose) polymerase 1 searches DNA via a 'monkey bar' mechanism.

Poly(ADP-ribose) polymerase 1 (PARP1) is both a first responder to DNA damage and a chromatin architectural protein. How PARP1 rapidly finds DNA damage sites in the context of a nucleus filled with undamaged DNA, to which it also binds, is an unresolved question. Here, we show that PARP1 association with DNA is diffusion-limited, and release of PARP1 from DNA is promoted by binding of an additional DNA molecule that facilitates a 'monkey bar' mechanism, also known as intersegment transfer. The WGR-domain of PARP1 is essential to this mechanism, and a point mutation (W589A) recapitulates the altered kinetics of the domain deletion. Demonstrating the physiological importance of the monkey bar mechanism for PARP1 function, the W589A mutant accumulates at sites of DNA damage more slowly following laser micro-irradiation than wild-type PARP1. Clinically relevant inhibitors of PARP1 did not alter the rate or mechanism of the release of PARP1 from DNA.

Dynamic behavior of DNA topoisomerase II\u03b2 in response to DNA double-strand breaks

DNA topoisomerase II (Topo II) is crucial for resolving topological problems of DNA and plays important roles in various cellular processes, such as replication, transcription, and chromosome segregation. Although DNA topology problems may also occur during DNA repair, the possible involvement of Topo II in this process remains to be fully investigated. Here, we show the dynamic behavior of human Topo IIβ in response to DNA double-strand breaks (DSBs), which is the most harmful form of DNA damage. Live cell imaging coupled with site-directed DSB induction by laser microirradiation demonstrated rapid recruitment of EGFP-tagged Topo IIβ to the DSB site. Detergent extraction followed by immunofluorescence showed the tight association of endogenous Topo IIβ with DSB sites. Photobleaching analysis revealed that Topo IIβ is highly mobile in the nucleus. The Topo II catalytic inhibitors ICRF-187 and ICRF-193 reduced the Topo IIβ mobility and thereby prevented Topo IIβ recruitment to DSBs. Furthermore, Topo IIβ knockout cells exhibited increased sensitivity to bleomycin and decreased DSB repair mediated by homologous recombination (HR), implicating the role of Topo IIβ in HR-mediated DSB repair. Taken together, these results highlight a novel aspect of Topo IIβ functions in the cellular response to DSBs.

Abstract 3218: Maternal embryonic leucine zipper kinase (MELK) confers radioresistance in triple-negative breast cancers (TNBC) through a nonhomologous end joining (NHEJ)-mediated pathway

Abstract Background: Increased rates of locoregional recurrence have been observed in TNBC despite the use of radiation (RT); therefore, approaches that result in radiosensitizaton in TNBC may improve clinical outcomes. Despite some controversy as to the role of MELK kinase as an oncogene, we previously established its clear role in conferring radioresistance in TNBC. Here we extend those studies to understand the mechanism of conferred radioresistance. Methods: Clonogenic survival assays were used to quantify the degree of radiosensitivity after MELK inhibition. Mass spectrometry was utilized to identify proteins associated with MELK. NHEJ assays were performed using linearized pEYFP vector and quantitated by FACS. qRT-PCR was used to determine MELK RNA expression. Rad51 and γH2AX foci counting were performed to evaluate homologous recombination (HR). Neutral comet assay was used to quantitate dsDNA break repair. Laser microirradiation studies were performed to determine the location and kinetics of protein shuttle to and from sites of dsDNA breaks, including MELK, Ku70 and Ku80 proteins. Fluorescent microscopy was performed for localization of endogenous MELK protein post RT. To study the effect of MELK kinase activity on Ku70-Ku80 complex formation and recruitment at the DNA damage site, cells were lysed and immunoprecipitation was performed using MELK, Ku70 or Ku80-specific antibodies. Results: We previously demonstrated that genomic or pharmacologic inhibition of MELK confers significant radiosensitization. Mechanistic studies were undertaken to understand the pathways critical to MELK-mediated radioresistance. When MELK is genomically or pharmacologically inhibited, NHEJ and HR reporter assays demonstrate that this radiosensitization is driven through impaired NHEJ at 4, 16, and 24 hrs after RT and not mediated by HR in multiple TNBC cell lines. Tandem mass spectrometry studies of tagged MELK protein identify Ku70 and Ku80 as direct interactors with MELK protein, which is confirmed by Co-IP. Laser microirradiation studies confirm that MELK, Ku70 and Ku80 co-localize to sites of dsDNA breaks, and that MELK kinase function is required to stabilize the Ku70/Ku80 complex at these sites. Comet assay confirms that rapid dissolution of the Ku70/Ku80 complex when MELK is inhibited or functionally dead is not a result of accelerated dsDNA break repair. Conclusion: MELK kinase function is critical in conferring radioresistance in TNBC and inhibition of function confers radiosensitivity through an NHEJ-mediated pathway. MELK also stabilizes Ku70/Ku80 proteins at sites of dsDNA breaks and allows for more efficient repair of breaks induced by ionizing RT. These results further support the rationale for developing clinical strategies to inhibit MELK in combination with RT treatment as a novel radiosensitizing strategy in TNBC. Citation Format: Shyam Nyati, Ben Chandler, Eric Olsen, Leah Moubadder, Meilan Liu, Meleah Cameron, Kari Wilder-Romans, Theodore S. Lawrence, Powel H. Brown, Fellix Y. Fang, Lori J. Pierce, Corey Speers. Maternal embryonic leucine zipper kinase (MELK) confers radioresistance in triple-negative breast cancers (TNBC) through a nonhomologous end joining (NHEJ)-mediated pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3218.

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation.

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (γ-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Microirradiation for Precise, Double-strand Break Induction in vivo in Caenorhabditis elegans.

DNA double-strand breaks (DSBs) are toxic lesions that every cell must accurately repair in order to survive. The repair of DSBs is an integral part of a cell life cycle and can lead to lethality if repaired incorrectly. Laser microirradiation is an established technique which has been used in yeast, mammalian cell culture, and Drosophila cell culture to study the regulation of DSB repair. Up to our studies, this method has not been adapted for use in a whole, live, multicellular organism to study this repair in vivo. We have recently shown that this system can be used for study of the recruitment of vital repair proteins to microirradiation-induced breaks in the transparent nematode Caenorhabditis elegans. With the integration of microirradiation and imaging technology, we can precisely induce DSBs in target nuclei and study the recruitment of fluorescently tagged repair proteins from the time of damage induction. Whole, live worms are plated and immobilized for targeting of nuclei, and immediately following induction the targeted region can be imaged for up to an hour and a half post-microirradiation. This method is the first that allows for study of DNA repair protein kinetics in vivo in an intact organism, which can be adapted in numerous ways to allow for study of repair kinetics in various aspects of the repair process.

Differential RPA-1 and RAD-51 recruitment in vivo throughout the C. elegans germline, as revealed by laser microirradiation.

Studies of the repair pathways associated with DNA double strand breaks (DSBs) are numerous, and provide evidence for cell-cycle specific regulation of homologous recombination (HR) by the regulation of its associated proteins. Laser microirradiation is a well-established method to examine in vitro kinetics of repair and allows for live-imaging of DSB repair from the moment of induction. Here we apply this method to whole, live organisms, introducing an effective system to analyze exogenous, microirradiation-induced breaks in the Caenorhabditis elegans germline. Through this method we observed the sequential kinetics of the recruitment of ssDNA binding proteins RPA-1 and RAD-51 in vivo. We analyze these kinetics throughout different regions of the germline, and thus throughout a range of developmental stages of mitotic and meiotic nuclei. Our analysis demonstrates a largely conserved timing of recruitment of ssDNA binding proteins to DSBs throughout the germline, with a delay of RAD-51 recruitment at mid-pachytene nuclei. Microirradiated nuclei are viable and undergo a slow kinetics of resolution. We observe RPA-1 and RAD-51 colocalization for hours post-microirradiation throughout the germline, suggesting that there are mixed RPA-1/RAD-51 filaments. Finally, through live imaging analysis we observed RAD-51 foci movement with low frequency of coalescence.

Abstract IA13: Combination of DNA methyltransferase and PARP inhibitors as a novel therapy strategy for multiple cancers: Key data in AML and triple negative breast cancer

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors represent one of the most exciting recent developments in cancer therapy. While substantial efficacy has been shown with clinically available PARP inhibitors (PARPis), to date, in treatment of hereditary deletions of BRCA1/2 in breast and ovarian cancers, the high promise of these drugs has not yet been realized in sporadic cancers. We present here strong preclinical data for a novel, mechanistically based, combinatorial approach to using DNA methyltransferase inhibitors (DNMTis), such as decitabine (DAC) and 5-Azacytidine (5-AZA), with PARP inhibitors (PARPis) as a treatment strategy for acute myelogenous leukemias (AML) and triple negative breast cancer (TNBC). We have previously demonstrated that low doses of 5-AZA and DAC alone show efficacy in AML and TNBC, and propose treatment with PARPis to enhance sensitivity of cancer cells to DNMTis. The mechanistic rationale for our approach is based upon: 1) data from our group and others showing DNMT1 and PARP1 associate in a complex, and this association increases with DNA damage; 2) the fact that 5-AZA and DAC trap DNMTs led us to hypothesize that these drugs might also increase PARP trapping at DNA damage sites; and 3) the cytotoxicity of the most potent PARPis (e.g. BMN 673) appears to correlate with the degree of trapping of PARP1 in chromatin. We first find that in cultured human AML and TNBC cells, the DNMTis (5 to 20 nM DAC or 100 to 200nM 5-AZA) and PARPis (1 to 10 nM BMN 673) alone trap PARP into chromatin, and this effect is enhanced when the drugs are combined. In addition, the PARPi-DNMTi combination treatment of TNBC cell line MDA-MB-231 resulted in significantly enhanced retention of PARP1 and DNMT1 at sites of double strand breaks (DSBs) induced by laser microirradiation. Concomitant with this, the combined doses resulted in significant increases in cytotoxic DSBs, observed 4-24 hours after DSB induction, when compared to single-drug treatments. Homologous recombination (HR) DSB repair activity also appears decreased, as measured by GFP reporter assays. In keeping with these findings, colony survival assays demonstrated that the combination treatment, compared to either drug alone, strongly inhibited colony formation of TNBC cell lines (N=4). Notably non-tumorigenic MCF10A cells showed no significant differences in colony numbers with single or combination drug treatments. Similar to TNBCs, AML cell lines (N=3) as well as primary AML cells (N=8) showed dramatic decreases in colonies in combination vs single agent drug treatments. In the most important translational implications of the preliminary studies, in in vivo therapy TNBC and AML models in immune-deficient mice, our low dose combinations of DNMTis and PARPis provide for potent anti-tumor responses. Mouse xenograft experiments using BRCA mutant TNBC cell line SUM149PT demonstrated that the combination treatment has a significant (p<0.05) survival advantage compared to control (vehicle), AZA (0.5mg/kg) or BMN (0.3 mg/kg) alone. Importantly, mouse MDA-MB-231 xenografts with intact BRCA1 showed significant survival with the combined drug treatment. Likewise in AML xenografts of MOLM14 and MV411 cell lines treated with the drug combination show significantly decrease leukemia burden, as measured by luciferase imaging. Our data suggest a novel use of both DNMTis and PARPis in a compelling therapeutic strategy for TNBCs independent of BRCA mutations and poor prognosis AML; the latter will be investigated in a clinical trial to be based at the University of Maryland and funded by Van Andel-SU2C. Citation Format: Nidal Muvarak, Khadiza Chowdhury, Carine Robert, Xia Limin, Eun Yong Choi, Yi Cai, Marina Bellani, Michael Seidman, Maria R. Baer, Rena Lapidus, Stephen B. Baylin, Feyruz V. Rassool. Combination of DNA methyltransferase and PARP inhibitors as a novel therapy strategy for multiple cancers: Key data in AML and triple negative breast cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA13.

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Protein phosphatase 1 and phosphatase 1 nuclear targeting subunit-dependent regulation of DNA-dependent protein kinase and non-homologous end joining.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a key role in mediating non-homologous end joining (NHEJ), a major repair pathway for DNA double-strand breaks (DSBs). The activation, function and dynamics of DNA-PKcs is regulated largely by its reversible phosphorylation at numerous residues, many of which are targeted by DNA-PKcs itself. Interestingly, these DNA-PKcs phosphorylation sites function in a distinct, and sometimes opposing manner, suggesting that they are differentially regulated via complex actions of both kinases and phosphatases. In this study we identified several phosphatase subunits as potential DSB-associated proteins. In particular, protein phosphatase 1 (PP1) is recruited to a DSB-mimicking substrate in Xenopus egg extracts and sites of laser microirradiation in human cells. Depletion of PP1 impairs NHEJ in both Xenopus egg extracts and human cells. PP1 binds multiple motifs of DNA-PKcs, regulates DNA-PKcs phosphorylation, and is required for DNA-PKcs activation after DNA damage. Interestingly, phosphatase 1 nuclear targeting subunit (PNUTS), an inhibitory regulator of PP1, is also recruited to DNA damage sites to promote NHEJ. PNUTS associates with the DNA-PK complex and is required for DNA-PKcs phosphorylation at Ser-2056 and Thr-2609. Thus, PNUTS and PP1 together fine-tune the dynamic phosphorylation of DNA-PKcs after DNA damage to mediate NHEJ.

Abstract 1404: Regulation of 53BP1 by the structural nuclear protein NuMA

Abstract Cells repair DNA double-strand breaks (DSB) through two major pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). The choice between these two pathways is critical to cell survival and is altered in cancers. 53BP1 is a protein with an important role in this choice: it promotes NHEJ by blocking CtIP-dependent DNA resection. Here, we present a new negative regulation mechanism of 53BP1 that relies on the structural protein NuMA. This protein participates in mitotic spindle assembly, influences chromatin organization during the interphase and modulates the chromatin response to DNA damage. We identified NuMA in a proteomics analysis of a 53BP1-interacting proteins. The interaction was confirmed by reciprocal immunoprecipitation and with a FRET assay. In response to DSB, we measured greater than 50% decreased 53BP1-NuMA binding, which suggests that NuMA may restrain 53BP1 diffusion in the absence of DNA damage. Fluorescence correlation spectroscopy (FCS) measurements in cells expressing GFP-tagged 53BP1 support this hypothesis. Moreover, NuMA overexpression inhibited 53BP1 recruitment at DNA damage sites in laser microirradiation assays, whereas NuMA silencing had the opposite effect. 53BP1 plays an essential role in B cell class switch recombination and mediates PARP inhibitor sensitivity in BRCA1-null cells. NuMA overexpression prevented immunoglobulin switching, and overwrote cancer cell sensitivity to PARP inhibitors to the same extent as 53BP1 loss of function. Our results help understand how 53BP1 is controlled, and shed light on the mechanisms regulating PARPi sensitivity. Citation Format: Naike Salvador Moreno. Regulation of 53BP1 by the structural nuclear protein NuMA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1404. doi:10.1158/1538-7445.AM2017-1404