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Artemis opens the hairpins generated in V(D)J recombination. V(D)J recombination occurs at sequences called 12-RSS and 23-RSS (triangles in the figure), where RSS designates recombination signal sequence. An RSS contains conserved heptamer and nonamer sequence elements, separated by either 12 or 23 non-conserved base pairs, and hence the designation 12-RSS and 23-RSS. One recombination event requires one 12-RSS and one 23-RSS, and this is called the 12/23 rule. In early lymphoid cells, the RAG complex (RAG1 and 2 along with the constitutively expressed HMGB1 protein) nicks and then hairpins the coding ends at the V and J segments in the figure. Ku can bind to any of the four DNA ends. The Artemis:DNA-PKcs complex then binds to the V and J hairpin ends and nicks the hairpins in a manner that usually results in a 3′ overhang. The ends can be processed further by the Artemis:DNA-PKcs complex and a DNA polymerase to introduce diversity. The NHEJ ligase complex then ligates the ends together. Some antigen receptor loci have not only V and J segments, but also D segments; hence, the name V(D)J recombination.
Source publication
Artemis is a vertebrate nuclease with both endo- and exonuclease activities that acts on a wide range of nucleic acid substrates.
It is the main nuclease in the non-homologous DNA end-joining pathway (NHEJ). Not only is Artemis important for the repair
of DNA double-strand breaks (DSBs) in NHEJ, it is essential in opening the DNA hairpin intermedia...
Contexts in source publication
Context 1
... located on the two strands within the duplex portion, directly adjacent to the double-to single-stranded DNA boundary. A third contact point (C, red arrowhead), which is also the catalytic site, is located 1-nt (or an equivalent dis- tance) on the 5 side of Contact point B (green dot) ( Figures 1 and 2); this catalytic site does not have to be on the same DNA strand as Contact point B (see Figure 1, 3 overhang substrate). In all cases, Artemis:DNA-PK cs is able to dis- tort the single-stranded portion of the substrate into a struc- ture resembling key features of the DNA hairpin substrate. ...
Context 2
... in this case, the 4th nt of this overhang is spatially located a distance equivalent to 1-nt 5 of Contact point B (∼3 to 5 angstroms). Thus, Artemis:DNA-PK cs ac- tivity on this substrate results in a 4-nt overhang at the 3 end ( Figure 1, 3 overhang substrate) (13). This model requires a polarity in the Artemis:DNA-PK cs complex in order to rec- ognize the helical pitch of the double-stranded DNA du- plex. ...
Context 3
... and signal ends that are blunt (Figure 3). The hairpin at the coding end must be opened to be joined to form a coding joint. ...
Context 4
... hairpin at the coding end must be opened to be joined to form a coding joint. Based on genetic and biochemical evidence, the hairpin opening at coding ends of the V, D and J segments during V(D)J recombination is completely dependent on the Artemis:DNA-PK cs complex ( Figure 3) (2,6). Since the Artemis:DNA-PK cs complex is located at each coding end at the time of hairpin opening, the cod- ing end resection is almost certainly due to Artemis:DNA- PK cs . ...
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B-lymphocytes in the bone marrow (BM) must generate a functional B-cell receptor and overcome the negative selection induced by reactivity with autoantigens. Two rounds of DNA recombination are required for the production of functional immunoglobulin heavy (Ig-HCs) and light (LCs) chains necessary for the continuation of B-lymphocyte development in...
Significance
Double-stranded DNA breaks pose a serious threat to the survival of cells. Nonhomologous end joining (NHEJ) is a crucial DNA repair pathway in which the DNA-dependent protein kinase (DNA-PK) complex, a key holoenzyme consisting of the Ku70/80 heterodimer and the catalytic subunit DNA-PKcs, senses DNA breaks and initiates the NHEJ repai...
Citations
... SNM1A-C have conserved b-CASP and metallo-b-lactamase (MBL) domains ( Fig. 1A-C). [6][7][8] The MBL fold is present in many hydrolases and other metalloenzymes, including Ambler class B Zn(II) dependent b-lactamases, where it has been shown that potent and selective in vivo inhibition can be achieved, 9,10 including as a result of optimising hits arising from a high-throughput screen. 11 Interestingly, given their central biological importance, there has been relatively little work on targeting MBL-fold nucleases, including SNM1A-C, as anti-cancer or other targets, with one exception being ongoing efforts to develop inhibitors of the mRNA MBL-fold processing Cleavage and Polyadenylation Specicity Factor (CPSF3) as a cancer target 12 and as an antiparasitic agent. ...
... We hypothesised that the carboxylic acid of K1 (6) may bind to one or both of the active-site metal ions. Indeed, when the carboxylic acid of 6 was replaced with normally less effective chelating groups, such as ester (7), amide (8) or hydroxyl (9) groups, there was a marked loss of SNM1A inhibition ( Fig. 3B and C). Given the potency of both 1 and 3 as SNM1A inhibitors and the information about binding provided by the SNM1A: 1 crystal structures (Fig. 2), we substituted the carboxylic acid of K1 (6) with a hydroxamic acid (10), a change which resulted in a 3-fold increase in potency ( Fig. 3B and C). ...
The three human SNM1 metallo-β-lactamase fold nucleases (SNM1A–C) play key roles in DNA damage repair and in maintaining telomere integrity. Genetic studies indicate that they are attractive targets for cancer treatment and to potentiate chemo- and radiation-therapy. A high-throughput screen for SNM1A inhibitors identified diverse pharmacophores, some of which were shown by crystallography to coordinate to the di-metal ion centre at the SNM1A active site. Structure and turnover assay-guided optimization enabled the identification of potent quinazoline–hydroxamic acid containing inhibitors, which bind in a manner where the hydroxamic acid displaces the hydrolytic water and the quinazoline ring occupies a substrate nucleobase binding site. Cellular assays reveal that SNM1A inhibitors cause sensitisation to, and defects in the resolution of, cisplatin-induced DNA damage, validating the tractability of MBL fold nucleases as cancer drug targets.
... These DNA breaks stimulate the cellular DNA repair processes, allowing site-specific genomic changes to be introduced more easily (Rouet et al., 1994;Choulika et al., 1995). Artemis' unique nuclease actions result in the formation of INDELs, rendering non-homologous end-joining repair systems inappropriate for precise alterations (Chang and Lieber, 2016). In situations where Homology-Directed Repair (HDR) is involved, a process that relies on a matching pair of chromosomes, the preservation of sequence information during the repair process is exceptionally high, exhibiting either minimal loss or no loss at all (known as the conservative type). ...
Crop improvement programmes began with traditional breeding practices since the inception of agriculture. Farmers and plant breeders continue to use these strategies for crop improvement due to their broad application in modifying crop genetic compositions. Nonetheless, conventional breeding has significant downsides in regard to effort and time. Crop productivity seems to be hitting a plateau as a consequence of environmental issues and the scarcity of agricultural land. Therefore, continuous pursuit of advancement in crop improvement is essential. Recent technical innovations have resulted in a revolutionary shift in the pattern of breeding methods, leaning further towards molecular approaches. Among the promising approaches, marker-assisted selection, QTL mapping, omics-assisted breeding, genome-wide association studies and genome editing have lately gained prominence. Several governments have progressively relaxed their restrictions relating to genome editing. The present review highlights the evolutionary and revolutionary approaches that have been utilized for crop improvement in a bid to produce climate-resilient crops observing the consequence of climate change. Additionally, it will contribute to the comprehension of plant breeding succession so far. Investing in advanced sequencing technologies and bioinformatics will deepen our understanding of genetic variations and their functional implications, contributing to breakthroughs in crop improvement and biodiversity conservation.
... The former were severely depleted in Prkdc or Polm deficient cells (LFC < -4 and LFC < -3, respectively), while the latter were mildly affected (LFC > -1, Figure 4d). This difference could be due to Prkdc-Dclre1c mediated 5' resection of the distal end of a blunt DSB, revealing 3' overhang substrate for Polm (see Discussion; 41 ). Consistent with this, PAM-. ...
The genome engineering capability of the CRISPR/Cas system depends on the DNA repair machinery to generate the final outcome. Several genes can have an impact on mutations created, but their exact function and contribution to the result of the repair are not completely characterised. This lack of knowledge has limited the ability to comprehend and regulate the editing outcomes. Here, we measure how the absence of 21 repair genes changes the mutation outcomes of Cas9-generated cuts at 2,812 synthetic target sequences in mouse embryonic stem cells. Absence of key non-homologous end joining genes Lig4, Xrcc4, and Xlf abolished small insertions and deletions, while disabling key microhomology-mediated repair genes Nbn and Polq reduced frequency of longer deletions. Complex alleles of combined insertion and deletions were preferentially generated in the absence of Xrcc6. We further discover finer structure in the outcome frequency changes for single nucleotide insertions and deletions between large microhomologies that are differentially modulated by the knockouts. We use the knowledge of the reproducible variation across repair milieus to build predictive models of Cas9 editing results that outperform the current standards. This work improves our understanding of DNA repair gene function, and provides avenues for more precise modulation of CRISPR/Cas9-generated mutations.
... The repair of DSBs occurs mainly in highly dense heterochromatin areas, such as the telomere [15,17]. Artemis can also promote p53 degradation by E3 ubiquitin ligase, and participate in apoptosis regulation and cell cycle progression by inhibiting p53 phosphorylation through DNA-PKcs [18,19]. In addition to repairing DNA damage directly, Artemis may also participate in regulating cell cycle checkpoint and signal transduction pathways. ...
Background:
Artemis belongs to the SNM1 gene family, and plays a role in repairing ionizing-radiation-induced DNA double-strand breaks and variable (diversity) joining recombination. S534, S538, S516, S645 represent four most rapid phosphorylation sites in Artemis, and serine phosphorylation at amino acid 516 is closely associated with activation. Artemis mutation is perceived as contributing to Omenn syndrome, which manifest features of severe combined immunodeficiency disease, associated with lymphadenopathy, hepatosplenomegaly, erythroderma and baldness. In addition, Artemis phosphorylated at serine 516 (Artemis S516-P) was expressed in scalp hair follicles (HF) as well as other skin appendages, and its expression level is important to mouse hair cycling. However, whether Artemis participated in the regulation of HF growth still unclear.
Methods:
Using immunofluorescence double-staining, we assessed the association between Artemis S516-P with proliferation, apoptosis, and differentiation markers in normal adult anagen scalp HF.
Results:
The results of double-staining immunofluorescence revealed overlapping expression pattern for Artemis S516-P and keratin16, similar pattern for c-myc and p21, while presenting opposite trends for keratin 10, phospho-p53, Bax, Bcl-2 and keratin 14.
Conclusions:
Our study provides the clues that Artemis may play roles in regulation of differentiation, proliferation, apoptosis and cell cycling during HF growth and development.
... In the resection step, DNA-PKcs forms a complex with the endonuclease Artemis and activates it immediately after undergoing autophosphorylation [33]. Thus, DNA sequence at the boundaries between ssDNA and dsDNA are resected by Artemis [34]. In addition to endonuclease activity on both the 5ʹ and the 3ʹ DNA overhangs, Artemis further exhibits an intrinsic 5' exonucleases activity on ssDNA even though absence of DNA-PKcs [35] (Fig. 1C). ...
Spermatogenesis is a complex developmental process. During this process, male germ cells from spermatogonia to sperm cells encounter a number of DNA damages. The most severe form of these damages is double-strand breaks (DSBs) deriving from exogenous and endogenous genotoxic insults. DSBs must be correctly repaired in a short time to maintain genomic integrity in the male germ cells. For this purpose, there are four pathways working in repaire of DSBs: homologous recombination (HR), classical non-homologous end joining (cNHEJ), alternative end joining (aEJ), and single strand annealing (SSA). While the HR pathway repairs DSBs with a homology-based and error-free manner, the cNHEJ, aEJ, and SSA pathways join free ends in a sequence-independent mechanism. Possible impairments in these DSB repair mechanisms can lead to cell cycle arrest, abnormal meiotic recombination, and ultimately male infertility. In this review, we comprehensively introduce DSB repair pathways being used by male germ cells during spermatogenesis. Also, potential relationship between dysfunction in these pathways and male infertility development are discussed in the light of existing studies.
... Artemis, which has intrinsic 5 to 3 exonuclease activity (21), acts as an endonuclease only when activated by DNA-PKcs (7). It can hydrolyze the phosphodiester bond at the junction of single-stranded to double-stranded DNA boundaries (22). At 5 overhangs, Artemis removes the overhang intact and leaves the dsDNA blunt and with a 5 P. At 3 overhangs, Artemis typically cuts 4 nts 3 of the boundary to leave a 4 nt 3 overhang. ...
Artemis nuclease and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are key components in nonhomologous DNA end joining (NHEJ), the major repair mechanism for double-strand DNA breaks. Artemis activation by DNA-PKcs resolves hairpin DNA ends formed during V(D)J recombination. Artemis deficiency disrupts development of adaptive immunity and leads to radiosensitive T- B- severe combined immunodeficiency (RS-SCID). An activated state of Artemis in complex with DNA-PK was solved by cryo-EM recently, which showed Artemis bound to the DNA. Here, we report that the pre-activated form (basal state) of the Artemis:DNA-PKcs complex is stable on an agarose-acrylamide gel system, and suitable for cryo-EM structural analysis. Structures show that the Artemis catalytic domain is dynamically positioned externally to DNA-PKcs prior to ABCDE autophosphorylation and show how both the catalytic and regulatory domains of Artemis interact with the N-HEAT and FAT domains of DNA-PKcs. We define a mutually exclusive binding site for Artemis and XRCC4 on DNA-PKcs and show that an XRCC4 peptide disrupts the Artemis:DNA-PKcs complex. All of the findings are useful in explaining how a hypomorphic L3062R missense mutation of DNA-PKcs could lead to insufficient Artemis activation, hence RS-SCID. Our results provide various target site candidates to design disruptors for Artemis:DNA-PKcs complex formation.
... Other nucleases also likely function in concert with CtIP/MRE11 to influence the extent of 3' ssDNA, and/or compete with CtIP to induce distinct end processing events [57]. Indeed, other nucleases have been implicated in end degradation during EJ, including Artemis, WRN, DNA2, FEN1, and Metnase [59,[61][62][63][64][65][66]. ...
... The Artemis nuclease appears to have a central role in processing DNA ends for C-NHEJ, particularly for opening the hairpin coding ends generated during V(D)J recombination [66]. In contrast to CtIP/MRE11-dependent end resection that favors formation of 3' ssDNA, Artemis can process DNA ends into distinct structures [66,67]. ...
... The Artemis nuclease appears to have a central role in processing DNA ends for C-NHEJ, particularly for opening the hairpin coding ends generated during V(D)J recombination [66]. In contrast to CtIP/MRE11-dependent end resection that favors formation of 3' ssDNA, Artemis can process DNA ends into distinct structures [66,67]. As examples, Artemis is proficient at processing 5' overhangs into blunt DNA ends, and blunt DNA ends into 3' overhangs [66,67]. ...
Chromosomal DNA double-strand breaks (DSBs) are the effective lesion of radiotherapy and other clastogenic cancer therapeutics, and are also the initiating event of many approaches to gene editing. Ligation of the DSBs by end joining (EJ) pathways can restore the broken chromosome, but the repair junctions can have insertion/deletion (indel) mutations. The indel patterns resulting from DSB EJ are likely defined by the initial structure of the DNA ends, how the ends are processed and synapsed prior to ligation, and the factors that mediate the ligation step. In this review, we describe key factors that influence these steps of DSB EJ in mammalian cells, which is significant both for understanding mutagenesis resulting from clastogenic cancer therapeutics, and for developing approaches to manipulating gene editing outcomes.
... The primary and abundant nuclease that is required is Artemis, which interacts with the FAT domain of DNA-PKcs and the N-terminus region of LIG4 [28][29][30][31]. Mentase and Artemis can trim 3 overhangs, but are more efficient later [32]. ...
In mammalian cells, double-strand breaks (DSBs) are repaired predominantly by error-prone non-homologous end joining (NHEJ), but less prevalently by error-free template-dependent homologous recombination (HR). DSB repair pathway selection is the bedrock for genome editing. NHEJ results in random mutations when repairing DSB, while HR induces high-fidelity sequence-specific variations, but with an undesirable low efficiency. In this review, we first discuss the latest insights into the action mode of NHEJ and HR in a panoramic view. We then propose the future direction of genome editing by virtue of these advancements. We suggest that by switching NHEJ to HR, full fidelity genome editing and robust gene knock-in could be enabled. We also envision that RNA molecules could be repurposed by RNA-templated DSB repair to mediate precise genetic editing.
... 26 Once bound, Ku70-Ku80 recruits DNA-PK, which in turn captures and phosphorylates Artemis, a protein with both 3 0 and 5 0 endonuclease activity. 28,29 Artemis prepares the DNA for ligation by trimming the overhanging strands into blunt ends. 28 Furthermore, DNA ends can be modified by the action of polymerases l and k into regions of microhomology (<4 nucleotides), which facilitate repair in certain types of damage. ...
... 28,29 Artemis prepares the DNA for ligation by trimming the overhanging strands into blunt ends. 28 Furthermore, DNA ends can be modified by the action of polymerases l and k into regions of microhomology (<4 nucleotides), which facilitate repair in certain types of damage. 30 Following trimming, a complex comprising PAXX, XRCC4, XRCC4-like factor, and DNA ligase IV (LIG4) is assembled, 31,32 after which LIG4 completes the process of repair by ligating the broken DNA strand. ...
The DNA damage response (DDR) is a complex set of downstream pathways triggered in response to DNA damage to maintain genomic stability. Many tumours exhibit mutations which inactivate components of the DDR, making them prone to the accumulation of DNA defects. These can both facilitate the development of tumours and provide potential targets for novel therapeutic interventions. The inhibition of the DDR has been shown to induce radiosensitivity in certain cancers, rendering them susceptible to treatment with radiotherapy and improving the therapeutic window. Moreover, DDR defects are a strong predictor of patient response to immune checkpoint inhibition (ICI). The ability to target the DDR selectively has the potential to expand the tumour neoantigen repertoire, thus increasing tumour immunogenicity and facilitating a CD8+ T and NK cell response against cancer cells. Combinatorial approaches, which seek to integrate DDR inhibition with radiotherapy and immunotherapy, have shown promise in early trials. Further studies are necessary to understand these synergies and establish reliable biomarkers.
... DNA-PK autophosphorylates DNA-PKcs to license further ligation activities (23,24). Artemis nuclease is essential for opening loops, bubbles, flaps, gaps, and hairpins, which are obstacles for ligation (25)(26)(27). Activation of Artemis requires both the DNA-PKcs protein and the kinase activity of either DNA-PKcs or ATM (24). XRCC4 and PAXX are requisite for the stabilization and activation of LIG4, which essentially ligates the DNA ends (28)(29)(30). ...
Non-homologous end joining (cNHEJ) is a major pathway to repair double-strand breaks (DSBs) in DNA. Several core cNHEJ are involved in the progress of the repair such as KU70 and 80, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Artemis, X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, and XRCC4-like factor (XLF). Recent studies have added a number of new proteins during cNHEJ. One of the newly identified proteins is Paralogue of XRCC4 and XLF (PAXX), which acts as a scaffold that is required to stabilize the KU70/80 heterodimer at DSBs sites and promotes the assembly and/or stability of the cNHEJ machinery. PAXX plays an essential role in lymphocyte development in XLF-deficient background, while XLF/PAXX double-deficient mouse embryo died before birth. Emerging evidence also shows a connection between the expression levels of PAXX and cancer development in human patients, indicating a prognosis role of the protein. This review will summarize and discuss the function of PAXX in DSBs repair and its potential role in cancer development.
