Role of 53BP1 in end protection and DNA synthesis at DNA breaks.

Double-strand break (DSB) repair choice is greatly influenced by the initial processing of DNA ends. 53BP1 limits the formation of recombinogenic single-strand DNA (ssDNA) in BRCA1-deficient cells, leading to defects in homologous recombination (HR). However, the exact mechanisms by which 53BP1 inhibits DSB resection remain unclear. Previous studies have identified two potential pathways: protection against DNA2/EXO1 exonucleases presumably through the Shieldin (SHLD) complex binding to ssDNA, and localized DNA synthesis through the CTC1-STN1-TEN1 (CST) and DNA polymerase α (Polα) to counteract resection. Using a combinatorial approach of END-seq, SAR-seq, and RPA ChIP-seq, we directly assessed the extent of resection, DNA synthesis, and ssDNA, respectively, at restriction enzyme-induced DSBs. We show that, in the presence of 53BP1, Polα-dependent DNA synthesis reduces the fraction of resected DSBs and the resection lengths in G0/G1, supporting a previous model that fill-in synthesis can limit the extent of resection. However, in the absence of 53BP1, Polα activity is sustained on ssDNA yet does not substantially counter resection. In contrast, EXO1 nuclease activity is essential for hyperresection in the absence of 53BP1. Thus, Polα-mediated fill-in partially limits resection in the presence of 53BP1 but cannot counter extensive hyperresection due to the loss of 53BP1 exonuclease blockade. These data provide the first nucleotide mapping of DNA synthesis at resected DSBs and provide insight into the relationship between fill-in polymerases and resection exonucleases.

MRI Is a DNA Damage Response Adaptor during Classical Non-homologous End Joining.

The modulator of retrovirus infection (MRI or CYREN) is a 30-kDa protein with a conserved N-terminal Ku-binding motif (KBM) and a C-terminal XLF-like motif (XLM). We show that MRI is intrinsically disordered and interacts with many DNA damage response (DDR) proteins, including the kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs and the classical non-homologous end joining (cNHEJ) factors Ku70, Ku80, XRCC4, XLF, PAXX, and XRCC4. MRI forms large multimeric complexes that depend on its N and C termini and localizes to DNA double-strand breaks (DSBs), where it promotes the retention of DDR factors. Mice deficient in MRI and XLF exhibit embryonic lethality at a stage similar to those deficient in the core cNHEJ factors XRCC4 or DNA ligase IV. Moreover, MRI is required for cNHEJ-mediated DSB repair in XLF-deficient lymphocytes. We propose that MRI is an adaptor that, through multivalent interactions, increases the avidity of DDR factors to DSB-associated chromatin to promote cNHEJ.

Recent advances in liposome development for studying protein-lipid interactions.

Protein-lipid interactions are crucial for various cellular biological processes like intracellular signaling, membrane transport, and cytoskeletal dynamics. Therefore, studying these interactions is essential to understand and unravel their specific functions. Nevertheless, the interacting proteins of many lipids are poorly understood and still require systematic study. Liposomes are the most well-known and familiar biomimetic systems used to study protein-lipid interactions. Although liposomes have been widely used for studying protein-lipid interactions in classical methods such as the co-flotation assay (CFA), co-sedimentation assay (CSA), and flow cytometric assay (FCA), an overview of their current applications and developments in high-throughput methods is not yet available. Here, we summarize the liposome development in low and high-throughput methods to study protein-lipid interactions. Besides, a constructive comment for each platform is presented to stimulate the advancement of these technologies in the future.

HCoDES reveals chromosomal DNA end structures with single-nucleotide resolution.

The structure of broken DNA ends is a critical determinant of the pathway used for DNA double-strand break (DSB) repair. Here, we develop an approach involving the hairpin capture of DNA end structures (HCoDES), which elucidates chromosomal DNA end structures at single-nucleotide resolution. HCoDES defines structures of physiologic DSBs generated by the RAG endonuclease, as well as those generated by nucleases widely used for genome editing. Analysis of G1 phase cells deficient in H2AX or 53BP1 reveals DNA ends that are frequently resected to form long single-stranded overhangs that can be repaired by mutagenic pathways. In addition to 3' overhangs, many of these DNA ends unexpectedly form long 5' single-stranded overhangs. The divergence in DNA end structures resolved by HCoDES suggests that H2AX and 53BP1 may have distinct activities in end protection. Thus, the high-resolution end structures obtained by HCoDES identify features of DNA end processing during DSB repair.

Omnidirectional, polarization-independent, ultra-broadband metamaterial perfect absorber using field-penetration and reflected-wave-cancellation.

In this work, we present the result of nickel (Ni)-based metamaterial perfect absorbers (MPA) with ultra-broadband close-to-one absorbance. The experimental broadband characteristic is significantly improved over the past effort on metamaterial perfect absorbers. An in-depth physical picture and quantitative analysis is presented to reveal the physical origin of its ultrabroadband nature. The key constituent is the cancellation of the reflected wave using ultra-thin, moderate-extinction metallic films. The ultra-thin metal thickness can reduce the reflection as the optical field penetrates through the metallic films. This leads to minimal reflection at each ultra-thin metal layer, and light is penetrating into the Ni/SiO2 stacking. More intuitively, when the layer thickness is much smaller than the photon wavelength, the layer is essentially invisible to the photons. This results in absorption in the metal thin-film through penetration while there is minimal reflection by the metal film. More importantly, the experimental evidence for omni-directionality and polarization-insensitivity are established for the proposed design. Detailed measurement is conducted. Due to the ultrathin metal layers and the satisfactory tolerance in dielectric thickness, the broadband absorption has minimal degradation at oblique incidence. Such a wide angle, polarization-insensitive, ultra-broadband MPA can be very promising in the future, and the optical physics using sub-skin-depth metal film can also facilitate miniaturized high-performance nano-photonic devices.

Mec1p associates with functionally compromised telomeres.

In many organisms, telomere DNA consists of simple sequence repeat tracts that are required to protect the chromosome end. In the yeast Saccharomyces cerevisiae, tract maintenance requires two checkpoint kinases of the ATM family, Tel1p and Mec1p. Previous work has shown that Tel1p is recruited to functional telomeres with shorter repeat tracts to promote telomerase-mediated repeat addition, but the role of Mec1p is unknown. We found that Mec1p telomere association was detected as cells senesced when telomere function was compromised by extreme shortening due to either the loss of telomerase or the double-strand break binding protein Ku. Exonuclease I effects the removal of the 5' telomeric strand, and eliminating it prevented both senescence and Mec1p telomere association. Thus, in contrast to Tel1p, Mec1p associates with short, functionally compromised telomeres.

Genetic approaches to aging in budding and fission yeasts: new connections and new opportunities.

Yeasts are powerful model systems to examine the evolutionarily conserved aspects of eukaryotic aging because they maintain many of the same core cellular signaling pathways and essential organelles as human cells. We constructed a strain of the budding yeast Saccharomyces cerevisiae that could monitor the distribution of proteins involved in heterochromatic silencing and aging, and isolated mutants that alter this distribution. The largest class of such mutants cause defects in mitochondrial function, and appear to cause changes in nuclear silencing separate from the well-known Rtg2p-dependent pathway that alters nuclear transcription in response to the loss of the mitochondrial genome. Mutants that inactivate the ATP2 gene, which encodes the ATPase subunit of the mitochondrial F(1)F(0)-ATPase, were isolated twice in our screen and identify a lifespan extending pathway in a gene that is conserved in both prokaryotes and eukaryotes. The budding yeast S. cerevisiae S. cerevisiae has been used with great success to identify other lifespan-extending pathways in screens using surrogate phenotypes such as stress resistance or silencing to identify random mutants, or in high throughput screens that utilize the deletion strain set resource. However, the direct selection of long-lived mutants from a pool of random mutants is more challenging. We have established a new chronological aging assay for the evolutionarily distant fission yeast Schizosaccharomyces pombe that recapitulates aspects of aging conserved in all eukaryotes. We have constructed a novel S. pombe S. pombe DNA insertion mutant bank, and used it to show that we can directly select for a long-lived mutant. The use of both the budding and fission yeast systems should continue to facilitate the identification and validation of lifespan extending pathways that are conserved in humans.

Ribosome biosynthesis and Hedgehog activity are cooperative actionable signaling mechanisms in breast cancer following radiotherapy.

Hyperactivated ribosome biosynthesis is attributed to a need for elevated protein synthesis that accommodates cell growth and division, and is characterized by nucleomorphometric alterations and increased nucleolar counts. Ribosome biogenesis is challenged when DNA-damaging treatments such as radiotherapy are utilized. Tumor cells that survive radiotherapy form the basis of recurrence, tumor progression, and metastasis. In order to survive and become metabolically revitalized, tumor cells need to reactivate RNA Polymerase I (RNA Pol I) to synthesize ribosomal RNA, an integral component of ribosomes. In this study, we showed that following radiation therapy, tumor cells from breast cancer patients demonstrate activation of a ribosome biosynthesis signature concurrent with enrichment of a signature of Hedgehog (Hh) activity. We hypothesized that GLI1 activates RNA Pol I in response to irradiation and licenses the emergence of a radioresistant tumor population. Our work establishes a novel role for GLI1 in orchestrating RNA Pol I activity in irradiated breast cancer cells. Furthermore, we present evidence that in these irradiated tumor cells, Treacle ribosome biogenesis factor 1 (TCOF1), a nucleolar protein that is important in ribosome biogenesis, facilitates nucleolar translocation of GLI1. Inhibiting Hh activity and RNA Pol I activity disabled the outgrowth of breast cancer cells in the lungs. As such, ribosome biosynthesis and Hh activity present as actionable signaling mechanisms to enhance the effectiveness of radiotherapy.

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe.

BACKGROUND: Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. RESULTS: An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. CONCLUSIONS: This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches.

A Flow Cytometry-Based Method for Analyzing DNA End Resection in G0- and G1-Phase Mammalian Cells.

DNA double strand breaks (DSBs) constantly arise in cells during normal cellular processes or upon exposure to genotoxic agents, and are repaired mostly by homologous recombination (HR) and non-homologous end joining (NHEJ). One key determinant of DNA DSB repair pathway choice is the processing of broken DNA ends to generate single strand DNA (ssDNA) overhangs, a process termed DNA resection. The generation of ssDNA overhangs commits DSB repair through HR and inhibits NHEJ. Therefore, DNA resection must be carefully regulated to avoid mis-repaired or persistent DSBs. Accordingly, many approaches have been developed to monitor ssDNA generation in cells to investigate genes and pathways that regulate DNA resection. Here we describe a flow cytometric approach measuring the levels of replication protein A (RPA) complex, a high affinity ssDNA binding complex composed of three subunits (RPA70, RPA32, and RPA14 in mammals), on chromatin after DNA DSB induction to assay DNA resection. This flow cytometric assay requires only conventional flow cytometers and can easily be scaled up to analyze a large number of samples or even for genetic screens of pooled mutants on a genome-wide scale. We adopt this assay in G0- and G1- phase synchronized cells where DNA resection needs to be kept in check to allow normal NHEJ.

A Whole Genome CRISPR/Cas9 Screening Approach for Identifying Genes Encoding DNA End-Processing Proteins.

DNA double-strand breaks (DSBs) are mainly repaired by homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of HR or NHEJ is dictated in part by whether the broken DNA ends are resected to generate extended single-stranded DNA (ssDNA) overhangs, which are quickly bound by the trimeric ssDNA binding complex RPA, the first step of HR. Here we describe a series of protocols for generating Abelson murine leukemia virus-transformed pre-B cells (abl pre-B cells) with stably integrated inducible Cas9 that can be used to identify and study novel pathways regulating DNA end processing. These approaches involve gene inactivation by CRISPR/Cas9, whole genome guide RNA (gRNA) library-mediated screen, and flow cytometry-based detection of chromatin-bound RPA after DNA damage.

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.

DNA-PK promotes DNA end resection at DNA double strand breaks in G0 cells.

DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs are predominately repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 murine and human cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in proliferating cells at the G1 or G2 phase of the cell cycle was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, which has important implications for DNA DSB repair in quiescent cells.

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

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

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

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

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity is required for V(D)J recombination.

A whole-genome CRISPR/Cas9 screen identified ATP2A2, the gene encoding the Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2 protein, as being important for V(D)J recombination. SERCAs are ER transmembrane proteins that pump Ca2+ from the cytosol into the ER lumen to maintain the ER Ca2+ reservoir and regulate cytosolic Ca2+-dependent processes. In preB cells, loss of SERCA2 leads to reduced V(D)J recombination kinetics due to diminished RAG-mediated DNA cleavage. SERCA2 deficiency in B cells leads to increased expression of SERCA3, and combined loss of SERCA2 and SERCA3 results in decreased ER Ca2+ levels, increased cytosolic Ca2+ levels, reduction in RAG1 and RAG2 gene expression, and a profound block in V(D)J recombination. Mice with B cells deficient in SERCA2 and humans with Darier disease, caused by heterozygous ATP2A2 mutations, have reduced numbers of mature B cells. We conclude that SERCA proteins modulate intracellular Ca2+ levels to regulate RAG1 and RAG2 gene expression and V(D)J recombination and that defects in SERCA functions cause lymphopenia.

Loss of H3K36 Methyltransferase SETD2 Impairs V(D)J Recombination during Lymphoid Development.

Repair of DNA double-stranded breaks (DSBs) during lymphocyte development is essential for V(D)J recombination and forms the basis of immunoglobulin variable region diversity. Understanding of this process in lymphogenesis has historically been centered on the study of RAG1/2 recombinases and a set of classical non-homologous end-joining factors. Much less has been reported regarding the role of chromatin modifications on this process. Here, we show a role for the non-redundant histone H3 lysine methyltransferase, Setd2, and its modification of lysine-36 trimethylation (H3K36me3), in the processing and joining of DNA ends during V(D)J recombination. Loss leads to mis-repair of Rag-induced DNA DSBs, especially when combined with loss of Atm kinase activity. Furthermore, loss reduces immune repertoire and a severe block in lymphogenesis as well as causes post-mitotic neuronal apoptosis. Together, these studies are suggestive of an important role of Setd2/H3K36me3 in these two mammalian developmental processes that are influenced by double-stranded break repair.

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

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

Deficiency of XLF and PAXX prevents DNA double-strand break repair by non-homologous end joining in lymphocytes.

Non-homologous end joining (NHEJ) is a major DNA double-strand break (DSB) repair pathway that functions in all phases of the cell cycle. NHEJ repairs genotoxic and physiological DSBs, such as those generated by ionizing radiation and during V(D)J recombination at antigen receptor loci, respectively. DNA end joining by NHEJ relies on the core factors Ku70, Ku80, XRCC4, and DNA Ligase IV. Additional proteins also play important roles in NHEJ. The XRCC4-like factor (XLF) participates in NHEJ through its interaction with XRCC4, and XLF deficiency in humans leads to immunodeficiency and increased sensitivity to ionizing radiation. However, XLF is dispensable for NHEJ-mediated DSB repair during V(D)J recombination in murine lymphocytes, where it may have redundant functions with other DSB repair factors. Paralog of XRCC4 and XLF (PAXX) is a recently identified NHEJ factor that has structural similarity to XRCC4 and XLF. Here we show that PAXX is also dispensable for NHEJ during V(D)J recombination and during the repair of genotoxic DSBs in lymphocytes. However, a combined deficiency of PAXX and XLF blocks NHEJ with a severity comparable to that observed in DNA Ligase IV-deficient cells. Similar to XLF, PAXX interacts with Ku through its C-terminal region, and mutations that disrupt Ku binding prevent PAXX from promoting NHEJ in XLF-deficient lymphocytes. Our findings suggest that the PAXX and XLF proteins may have redundant functions during NHEJ.

A fully functionalized metamaterial perfect absorber with simple design and implementation.

Broadband perfect metamaterial absorbers have been drawing significant attention in recent years. A close-to-unity absorption over a broad spectral range is established and this facilitates many photonic applications. A more challenging goal is to construct a broadband absorber with a tailored spectral absorption. The spectral absorption control and spectral shaping are very critical in many applications, such as thermal-photovoltaic, thermal emitters, spectrum imaging system, biomedical and extraterrestrial sensing, and refractive index sensor. In this work, one-dimensional (1D) planar stacking structure is designed to achieve the ultimate goal of a functionalized absorber with a fully tailorable spectral absorption. The lithography and etching process are totally eliminated in this proposed structure, and the fabrication is fully compatible with the regular silicon IC processing. By using ~2 nm ultra-thin metallic layers with a 10-pair (10X) SiO2/Si3N4 integrated dielectric filter, we can achieve decent spectral response shaping. The planar configuration of the ultra-thin-metal metamaterial perfect absorber (MPA) is the key to the easy design/integration of the dielectric filters on top of the MPA. Specifically, band-rejected, high-pass, low-pass and band-pass structure are constructed successfully. Finally, experimental evidence to support our simulation result is also provided, which proves the feasibility of our proposal.