DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage because they can result in the loss of large chromosomal regions. In all mammalian cells, DSBs that occur throughout the cell ...cycle are repaired predominantly by the non-homologous DNA end joining (NHEJ) pathway. Defects in NHEJ result in sensitivity to ionizing radiation and the ablation of lymphocytes. The NHEJ pathway utilizes proteins that recognize, resect, polymerize and ligate the DNA ends in a flexible manner. This flexibility permits NHEJ to function on a wide range of DNA-end configurations, with the resulting repaired DNA junctions often containing mutations. In this Review, we discuss the most recent findings regarding the relative involvement of the different NHEJ proteins in the repair of various DNA-end configurations. We also discuss the shunting of DNA-end repair to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA) and the relevance of these different pathways to human disease.
DNA double-strand breaks (DSBs) are common lesions that continually threaten genomic integrity. Failure to repair a DSB has deleterious consequences, including cell death. Misrepair is also fraught ...with danger, especially inappropriate end-joining events, which commonly underlie oncogenic transformation and can scramble the genome. Canonically, cells employ two basic mechanisms to repair DSBs: homologous recombination (HR) and the classical nonhomologous end-joining pathway (cNHEJ). More recent experiments identified a highly error-prone NHEJ pathway, termed alternative NHEJ (aNHEJ), which operates in both cNHEJ-proficient and cNHEJ-deficient cells. aNHEJ is now recognized to catalyze many genome rearrangements, some leading to oncogenic transformation. Here, we review the mechanisms of cNHEJ and aNHEJ, their interconnections with the DNA damage response (DDR), and the mechanisms used to determine which of the three DSB repair pathways is used to heal a particular DSB. We briefly review recent clinical applications involving NHEJ and NHEJ inhibitors.
Inactivation of the retinoblastoma tumor suppressor gene (
) leads to genome instability, and can be detected in retinoblastoma and other cancers. One damaging effect is causing DNA double strand ...breaks (DSB), which, however, can be repaired by homologous recombination (HR), classical non-homologous end joining (C-NHEJ), and micro-homology mediated end joining (MMEJ). We aimed to study the mechanistic roles of RB in regulating multiple DSB repair pathways. Here we show that HR and C-NHEJ are decreased, but MMEJ is elevated in RB-depleted cells. After inducing DSB by camptothecin, RB co-localizes with CtIP, which regulates DSB end resection. RB depletion leads to less RPA and native BrdU foci, which implies less end resection. In RB-depleted cells, less CtIP foci, and a lack of phosphorylation on CtIP Thr847, are observed. According to the synthetic lethality principle, based on the altered DSB repair pathway choice, after inducing DSBs by camptothecin, RB depleted cells are more sensitive to co-treatment with camptothecin and MMEJ blocker poly-ADP ribose polymerase 1 (PARP1) inhibitor. We propose a model whereby RB can regulate DSB repair pathway choice by mediating the CtIP dependent DNA end resection. The use of PARP1 inhibitor could potentially improve treatment outcomes for RB-deficient cancers.
Sirtuins, a family of protein deacetylases, promote cellular homeostasis by mediating communication between cells and environment. The enzymatic activity of the mammalian sirtuin SIRT7 targets ...acetylated lysine in the N‐terminal tail of histone H3 (H3K18Ac), thus modulating chromatin structure and transcriptional competency. SIRT7 deletion is associated with reduced lifespan in mice through unknown mechanisms. Here, we show that SirT7‐knockout mice suffer from partial embryonic lethality and a progeroid‐like phenotype. Consistently, SIRT7‐deficient cells display increased replication stress and impaired DNA repair. SIRT7 is recruited in a PARP1‐dependent manner to sites of DNA damage, where it modulates H3K18Ac levels. H3K18Ac in turn affects recruitment of the damage response factor 53BP1 to DNA double‐strand breaks (DSBs), thereby influencing the efficiency of non‐homologous end joining (NHEJ). These results reveal a direct role for SIRT7 in DSB repair and establish a functional link between SIRT7‐mediated H3K18 deacetylation and the maintenance of genome integrity.
Synopsis
Sirtuins are deacetylase enzymes implicated in genome stability and life span regulation. Here, the roles of SIRT7 in genome function are studied by characterization of SirT7−/− mice.
Loss of SIRT7 results in reduced embryonic viability, accelerated aging, and genomic instability.
SIRT7 is recruited to sites of DNA damage in a PARP‐dependent manner.
SIRT7 participates in the DNA damage response by promoting NHEJ DNA repair.
SIRT7‐dependent H3K18 deacetylation is important for efficient 53BP1 recruitment at DNA damage sites.
SIRT7‐deficient cells show increased levels of replication stress, which may underlie progeroid phenotypes.
Increased replication stress and impaired DNA damage response in SIRT7‐deficient cells are linked to developmental defects and accelerated again in SIRT7 KO mice.
DNA double‐strand breaks are a threat to genome integrity and cell viability. The nucleolytic processing of broken DNA ends plays a central role in dictating the repair processes that will mend these ...lesions. Usually, DNA end resection promotes repair by homologous recombination, whereas minimally processed ends are repaired by non‐homologous end joining. Important in this process is the chromatin‐binding protein 53BP1, which inhibits DNA end resection. How 53BP1 shields DNA ends from nucleases has been an enduring mystery. The recent discovery of shieldin, a four‐subunit protein complex with single‐stranded DNA‐binding activity, illuminated a strong candidate for the ultimate effector of 53BP1‐dependent end protection. Shieldin consists of REV7, a known 53BP1‐pathway component, and three hitherto uncharacterized proteins: C20orf196 (SHLD1), FAM35A (SHLD2), and CTC‐534A2.2 (SHLD3). Shieldin promotes many 53BP1‐associated activities, such as the protection of DNA ends, non‐homologous end joining, and immunoglobulin class switching. This review summarizes the identification of shieldin and the various models of shieldin action and highlights some outstanding questions requiring answers to gain a full molecular understanding of shieldin function.
How 53BP1 shields DNA ends from resection has been a mystery. This review summarizes the recent discovery of the protein complex shieldin, and how it promotes 53BP1 activities, such as DNA end protection, NHEJ and Ig class switching.
According to the development trend of modern car body, the future car body will be composed of steel, aluminum‐magnesium alloy, plastic, carbon fiber reinforced polymer (CFRP), and other lightweight ...materials. Hybrid material body represents the latest development trend of car body structure in the future. With the development of lightweight technology, the application of carbon fiber composite material in automobile bodies is increasing due to its exceptional performance. Therefore, the joining of carbon fiber sheets with dissimilar sheets process in the hybrid material body is a key technical problem that needs to be solved. In this paper, the existing joining processes of adhesive process, mechanical joining, welding process, and hybrid joining for carbon fiber composites are reviewed; the principle, development, advantages, and shortcomings of each process for carbon fiber composites joining processes are systematically described; and the latest progress of the joining process are introduced. The purpose is tantamount to broaden the application range of carbon fiber sheets with dissimilar sheets and provide reference for its extensive application in the development of hybrid car body and lightweight.
Laser transmission joining (LTJ) is growing in importance, and has the potential to become a niche technique for the fabrication of hybrid plastic-metal joints for medical device applications. The ...possibility of directly joining plastics to metals by LTJ has been demonstrated by a number of recent studies. However, a reliable and quantitative method for defining the contact area between the plastic and metal, facilitating calculation of the mechanical shear stress of the hybrid joints, is still lacking. A new method, based on image analysis using ImageJ, is proposed here to quantify the contact area at the joint interface. The effect of discolouration on the mechanical performance of the hybrid joints is also reported for the first time. Biocompatible polyethylene terephthalate (PET) and commercially pure titanium (Ti) were selected as materials for laser joining using a 200W CW fibre laser system. The effect of laser power, scanning speed and stand-off distance between the nozzle tip and top surface of the plastic were studied and analysed by Taguchi L9 orthogonal array and ANOVA respectively. The surface morphology, structure and elemental composition on the PET and Ti surfaces after shearing/peeling apart were characterized by SEM, EDX, XRD and XPS.
Display omitted
•Laser transmission welding (LTW) is a fast, clean and adhesive-free method for direct joining between plastics and metals.•The true bond area between the contacting plastic and metal surface is quantified using a novel image analysis method.•SEM, EDX, XRD and XPS were used to produce a model for the cross-section of the bond region.•The locus of failure was through unmodified PET close to the laser modified carbon-rich layer on the Ti surface.•The method and results can be used in future research to improve the integrity of laser transmission welded joints.
Cells employ potentially mutagenic DNA repair mechanisms to avoid the detrimental effects of chromosome breaks on cell survival. While classical non‐homologous end‐joining (cNHEJ) is largely ...error‐free, alternative end‐joining pathways have been described that are intrinsically mutagenic. Which end‐joining mechanisms operate in germ and embryonic cells and thus contribute to heritable mutations found in congenital diseases is, however, still largely elusive. Here, we determined the genetic requirements for the repair of CRISPR/Cas9‐induced chromosomal breaks of different configurations, and establish the mutational consequences. We find that cNHEJ and polymerase theta‐mediated end‐joining (TMEJ) act both parallel and redundant in mouse embryonic stem cells and account for virtually all end‐joining activity. Surprisingly, mutagenic repair by polymerase theta (Pol θ, encoded by the Polq gene) is most prevalent for blunt double‐strand breaks (DSBs), while cNHEJ dictates mutagenic repair of DSBs with protruding ends, in which the cNHEJ polymerases lambda and mu play minor roles. We conclude that cNHEJ‐dependent repair of DSBs with protruding ends can explain de novo formation of tandem duplications in mammalian genomes.
Synopsis
Embryonic stem cells utilize two distinct mutagenic end‐joining mechanisms to repair chromosomal breaks. The configuration of the break dictates repair‐pathway choice, resulting in different repair outcomes, which can explain mutational signatures observed in human disease and evolution.
Theta‐mediated end‐joining (TMEJ) acts as a first line defense mechanism for chromosomal breaks in mouse embryonic stem cells.
Mutational signatures obtained in various human disease can be explained by TMEJ‐dependent repair of chromosomal breaks.
Classical non‐homologous end‐joining (cNHEJ) of chromosomal breaks with protruding ends can explain the formation of tandem duplications in mammalian genomes.
The introduction of one single chromosomal break induces lethality in cells that lack both TMEJ and cNHEJ.
DNA double‐strand break configuration dictates the choice between two distinct error‐prone repair pathways in mouse embryonic stem cells, resulting in different repair outcomes.
The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting ...event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals variable and often slow repair rates, with half-life times up to ∼10 hr. Furthermore, repair of the DSBs tends to be error prone. Both classical and microhomology-mediated end joining pathways contribute to the erroneous repair. Estimation of their individual rate constants indicates that the balance between these two pathways changes over time and can be altered by additional ionizing radiation. Our approach provides quantitative insights into DSB repair kinetics and fidelity in single loci and indicates that Cas9-induced DSBs are repaired in an unusual manner.
Display omitted
•Approach to measure single-locus DSB repair kinetics after Cas9-induced breaks•Multiple repair pathways can act on a single locus with distinct kinetics•Repair tends to be slow and error prone, although this depends on the locus
Brinkman et al. report a strategy to determine the rate and fidelity of double-strand break repair at single loci cut by Cas9. They also infer the contributions from different repair pathways. Cas9-induced breaks are repaired at variable but often slow rates, and the repair tends to be error prone.
PDF
