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42-613: NHEJ is typically guided by short homologous DNA sequences called microhomologies. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, hallmarks of tumor cells. NHEJ implementations are understood to have been existent throughout nearly all biological systems and it

84-405: A base pair . Blunt ends are not always desired in biotechnology since when using a DNA ligase to join two molecules into one, the yield is significantly lower with blunt ends. When performing subcloning, it also has the disadvantage of potentially inserting the insert DNA in the opposite orientation desired. On the other hand, blunt ends are always compatible with each other. Here is an example of

126-538: A 3' overhang by some DNA polymerases . Most commonly this is used in cloning PCR products created by such an enzyme. The product is joined with a linear DNA molecule with a 3' thymine overhang. Since adenine and thymine form a base pair , this facilitates the joining of the two molecules by a ligase, yielding a circular molecule. Here is an example of an A-overhang: Longer overhangs are called cohesive ends or sticky ends . They are most often created by restriction endonucleases when they cut DNA. Very often they cut

168-467: A complex with DNA-PKcs, which is present in mammals but absent in yeast . Ku is a basket-shaped molecule that slides onto the DNA end and translocates inward. Ku may function as a docking site for other NHEJ proteins, and is known to interact with the DNA ligase IV complex and XLF . End processing involves removal of damaged or mismatched nucleotides by nucleases and resynthesis by DNA polymerases. This step

210-456: A few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 ( Cdc28 in yeast), which is turned off in G1 and expressed in S and G2 . Cdk1 phosphorylates

252-523: A homodimeric Ku, but the three functions of LigD are broken up into three single-domain proteins sharing an operon. All three genes retain substantial homology with their LigD counterparts and the polymerase retains the preference for rNTP. NHEJ has been lost and acquired multiple times in bacteria and archaea, with a significant amount of horizontal gene transfer shuffling the system around taxa. Corndog and Omega, two related mycobacteriophages of Mycobacterium smegmatis , also encode Ku homologs and exploit

294-432: A number of bacteria, including Bacillus subtilis , Mycobacterium tuberculosis , and Mycobacterium smegmatis . Bacteria utilize a remarkably compact version of NHEJ in which all of the required activities are contained in only two proteins: a Ku homodimer and the multifunctional ligase/polymerase/nuclease LigD . In mycobacteria, NHEJ is much more error prone than in yeast, with bases often added to and deleted from

336-486: A process known as blunting, which involves filling in the sticky end with complementary nucleotides. This yields a blunt end, however, sticky ends are often preferable, meaning the main use of this method is to label DNA by using radiolabeled nucleotides to fill the gap. Blunt ends can also be converted to sticky ends by addition of double-stranded linker sequences containing recognition sequences for restriction endonucleases that create sticky ends and subsequent application of

378-453: A region of a double stranded (or other multi-stranded) DNA molecule near the end with a significant proportion of non-complementary sequences; that is, a sequence where nucleotides on the adjacent strands do not match up correctly: The term "frayed" is used because the incorrectly matched nucleotides tend to avoid bonding, thus appearing similar to the strands in a fraying piece of rope. Although non-complementary sequences are also possible in

420-409: A small piece of blunt-ended DNA: Non-blunt ends are created by various overhangs . An overhang is a stretch of unpaired nucleotides in the end of a DNA molecule. These unpaired nucleotides can be in either strand, creating either 3' or 5' overhangs. These overhangs are in most cases palindromic. The simplest case of an overhang is a single nucleotide. This is most often adenine and is created as

462-620: Is also required for subtelomeric silencing, the process by which genes located near telomeres are turned off. Several human syndromes are associated with dysfunctional NHEJ. Hypomorphic mutations in LIG4 and XLF cause LIG4 syndrome and XLF-SCID, respectively. These syndromes share many features including cellular radiosensitivity, microcephaly and severe combined immunodeficiency (SCID) due to defective V(D)J recombination . Loss-of-function mutations in Artemis also cause SCID, but these patients do not show

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504-409: Is because the primer terminus used to initiate DNA synthesis is less stable at 3' overhangs, necessitating a specialized NHEJ polymerase. The DNA ligase IV complex, consisting of the catalytic subunit DNA ligase IV and its cofactor XRCC4 (Dnl4 and Lif1 in yeast), performs the ligation step of repair. XLF , also known as Cernunnos, is homologous to yeast Nej1 and is also required for NHEJ. While

546-520: Is encoded by the NHEJ1 gene . XLF was originally discovered as the protein mutated in five patients with growth retardation, microcephaly, and immunodeficiency. The protein is required for the non-homologous end joining (NHEJ) pathway of DNA repair . Patients with XLF mutations also have immunodeficiency due to a defect in V(D)J recombination , which uses NHEJ to generate diversity in the antibody repertoire of

588-413: Is essential for viability in mammals. In contrast, mice lacking Ku or DNA-PKcs are viable, probably because low levels of end joining can still occur in the absence of these components. All NHEJ mutant mice show a SCID phenotype, sensitivity to ionizing radiation, and neuronal apoptosis. A system was developed for measuring NHEJ efficiency in the mouse. NHEJ efficiency could be compared across tissues of

630-441: Is longer than the other (typically by at least a few nucleotides), such that the longer strand has bases which are left unpaired. In blunt ends , both strands are of equal length – i.e. they end at the same base position, leaving no unpaired bases on either strand. The concept is used in molecular biology , in cloning , or when subcloning insert DNA into vector DNA . Such ends may be generated by restriction enzymes that break

672-476: Is not dependent on HR and NHEJ. These results showed that the repair mechanism of HLS is independent of NHEJ and HR pathways The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even

714-727: Is not necessary if the ends are already compatible and have 3' hydroxyl and 5' phosphate termini. Little is known about the function of nucleases in NHEJ. Artemis is required for opening the hairpins that are formed on DNA ends during V(D)J recombination , a specific type of NHEJ, and may also participate in end trimming during general NHEJ. Mre11 has nuclease activity, but it seems to be involved in homologous recombination , not NHEJ. The X family DNA polymerases Pol λ and Pol μ (Pol4 in yeast ) fill gaps during NHEJ. Yeast lacking Pol4 are unable to join 3' overhangs that require gap filling, but remain proficient for gap filling at 5' overhangs. This

756-414: Is possible to create a plasmid by excising a piece of DNA (using a different enzyme for each end) and then joining it to another DNA molecule with ends trimmed by the same enzymes. Since the overhangs have to be complementary in order for the ligase to work, the two molecules can only join in one orientation. This is often highly desirable in molecular biology . Sticky ends can be converted to blunt ends by

798-430: Is recruited to DSBs early and is thought to promote bridging of the DNA ends. The corresponding mammalian complex of Mre11-Rad50- Nbs1 ( MRN ) is also involved in NHEJ, but it may function at multiple steps in the pathway beyond simply holding the ends in proximity. DNA-PKcs is also thought to participate in end bridging during mammalian NHEJ. Eukaryotic Ku is a heterodimer consisting of Ku70 and Ku80 , and forms

840-455: Is required for the transcription of Nej1. NHEJ and heat-labile sites Induction of heat-labile sites (HLS) is a signature of ionizing radiation. The DNA clustered damage sites consist of different types of DNA lesions. Some of these lesions are not prompt DSBs but they convert to DSB after heating. HLS are not evolved to DSB under physiological temperature (37 C). Also, the interaction of HLS with other lesions and their role in living cells

882-799: Is the most favorable outcome , error-prone repair in V(D)J recombination is beneficial in that it maximizes diversity in the coding sequence of these genes. Patients with mutations in NHEJ genes are unable to produce functional B cells and T cells and suffer from severe combined immunodeficiency (SCID). Telomeres are normally protected by a "cap" that prevents them from being recognized as double-strand breaks. Loss of capping proteins causes telomere shortening and inappropriate joining by NHEJ, producing dicentric chromosomes which are then pulled apart during mitosis. Paradoxically, some NHEJ proteins are involved in telomere capping. For example, Ku localizes to telomeres and its deletion leads to shortened telomeres. Ku

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924-474: Is the predominant double-strand break repair pathway in mammalian cells. In budding yeast ( Saccharomyces cerevisiae ), however, homologous recombination dominates when the organism is grown under common laboratory conditions. When the NHEJ pathway is inactivated, double-strand breaks can be repaired by a more error-prone pathway called microhomology-mediated end joining (MMEJ). In this pathway, end resection reveals short microhomologies on either side of

966-424: Is yet elusive. The repair mechanisms of these sites are not fully revealed. The NHEJ is the dominant DNA repair pathway throughout the cell cycle. The DNA-PKcs protein is the critical element in the center of NHEJ. Using DNA-PKcs KO cell lines or inhibition of DNA-PKcs does not affect the repair capacity of HLS. Also blocking both HR and NHEJ repair pathways by dactolisib (NVP-BEZ235) inhibitor showed that repair of HLS

1008-531: The ATM kinase causes a synthetic defect in NHEJ, suggesting partial redundancy in the function of these two proteins in mice. XLF is structurally similar to XRCC4 , existing as a constitutive dimer with an N-terminal globular head domain, an alpha-helical stalk, and an unstructured C-terminal region (CTR). XLF has been shown to interact with XRCC4 , and with Ku protein , and it can also interact weakly with DNA. Co-crystal structures of XLF and XRCC4 suggest that

1050-518: The Artemis nuclease and joined by NHEJ. A specialized DNA polymerase called terminal deoxynucleotidyl transferase (TdT), which is only expressed in lymph tissue, adds nontemplated nucleotides to the ends before the break is joined. This process couples "variable" (V), "diversity" (D), and "joining" (J) regions, which when assembled together create the variable region of a B-cell or T-cell receptor gene. Unlike typical cellular NHEJ, in which accurate repair

1092-434: The 3' end and the 5' end (usually pronounced "three prime end" and "five prime end"). The numbers refer to the numbering of carbon atoms in the deoxyribose , which is a sugar forming an important part of the backbone of the DNA molecule. In the backbone of DNA the 5' carbon of one deoxyribose is linked to the 3' carbon of another by a phosphodiester bond linkage. When a molecule of DNA is double stranded, as DNA usually is,

1134-480: The NHEJ pathway to recircularize their genomes during infection. Unlike homologous recombination, which has been studied extensively in bacteria, NHEJ was originally discovered in eukaryotes and was only identified in prokaryotes in the past decade. In contrast to bacteria, NHEJ in eukaryotes utilizes a number of proteins , which participate in the following steps: In yeast, the Mre11-Rad50-Xrs2 ( MRX ) complex

1176-539: The break, which are then aligned to guide repair. This contrasts with classical NHEJ, which typically uses microhomologies already exposed in single-stranded overhangs on the DSB ends. Repair by MMEJ therefore leads to deletion of the DNA sequence between the microhomologies. Many species of bacteria, including Escherichia coli , lack an end joining pathway and thus rely completely on homologous recombination to repair double-strand breaks. NHEJ proteins have been identified in

1218-521: The ends of double-strand breaks during repair. Many of the bacteria that possess NHEJ proteins spend a significant portion of their life cycle in a stationary haploid phase, in which a template for recombination is not available. NHEJ may have evolved to help these organisms survive DSBs induced during desiccation. It preferentially use rNTPs (RNA nucleotides), possibly advantageous in dormant cells. The archaeal NHEJ system in Methanocella paludicola have

1260-405: The immune system. XLF interacts with DNA ligase IV and XRCC4 and is thought to be involved in the end-bridging or ligation steps of NHEJ. The yeast ( Saccharomyces cerevisiae ) homolog of XLF is Nej1 . In contrast to the profound immunodeficiency phenotype of XLF deletion in humans, deletion of XLF alone has a mild phenotype in mice. However, combining a deletion of XLF with deletion of

1302-488: The level of NHEJ protein Ku80 in human, cow, and mouse indicated that Ku80 levels vary dramatically between species, and that these levels are strongly correlated with species longevity. DNA end DNA ends refer to the properties of the ends of linear DNA molecules, which in molecular biology are described as "sticky" or "blunt" based on the shape of the complementary strands at the terminus. In sticky ends , one strand

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1344-597: The middle of double stranded DNA, mismatched regions away from the ends are not referred to as "frayed". Ronald W. Davis first discovered sticky ends as the product of the action of EcoRI , the restriction endonuclease . Sticky end links are different in their stability. Free energy of formation can be measured to estimate stability. Free energy approximations can be made for different sequences from data related to oligonucleotide UV thermal denaturation curves. Also predictions from molecular dynamics simulations show that some sticky end links are much stronger in stretch than

1386-514: The molecule's phosphodiester backbone at specific locations, which themselves belong to a larger class of enzymes called exonucleases and endonucleases . A restriction enzyme that cuts the backbones of both strands at non-adjacent locations leaves a staggered cut, generating two overlapping sticky ends, while an enzyme that makes a straight cut (at locations directly across from each other on both strands) generates two blunt ends. A single-stranded non-circular DNA molecule has two non-identical ends,

1428-649: The neurological defects associated with LIG4 or XLF mutations. The difference in severity may be explained by the roles of the mutated proteins. Artemis is a nuclease and is thought to be required only for repair of DSBs with damaged ends, whereas DNA Ligase IV and XLF are required for all NHEJ events. Mutations in genes that participate in non-homologous end joining lead to ataxia-telangiectasia (ATM gene) , Fanconi anemia (multiple genes), as well as hereditary breast and ovarian cancers (BRCA1 gene). Many NHEJ genes have been knocked out in mice . Deletion of XRCC4 or LIG4 causes embryonic lethality in mice, indicating that NHEJ

1470-462: The nuclease Sae2, allowing resection to initiate. NHEJ plays a critical role in V(D)J recombination , the process by which B-cell and T-cell receptor diversity is generated in the vertebrate immune system . In V(D)J recombination, hairpin-capped double-strand breaks are created by the RAG1/RAG2 nuclease , which cleaves the DNA at recombination signal sequences. These hairpins are then opened by

1512-405: The others. XLF Protein 2QM4 , 2R9A , 3Q4F , 3RWR , 3SR2 , 3W03 79840 75570 ENSG00000187736 ENSMUSG00000026162 Q9H9Q4 Q3KNJ2 NM_024782 NM_029342 NP_079058 NP_001364427 NP_001364428 NP_083618 Non-homologous end-joining factor 1 (NHEJ1), also known as Cernunnos or XRCC4-like factor ( XLF ), is a protein that in humans

1554-468: The precise role of XLF is unknown, it interacts with the XRCC4/DNA ligase IV complex and likely participates in the ligation step. Recent evidence suggests that XLF promotes re-adenylation of DNA ligase IV after ligation, recharging the ligase and allowing it to catalyze a second ligation. In yeast, Sir2 was originally identified as an NHEJ protein, but is now known to be required for NHEJ only because it

1596-526: The restriction enzyme or by homopolymer tailing, which refers to extending the molecule's 3' ends with only one nucleotide, allowing for specific pairing with the matching nucleotide (e.g. poly-C with poly-G). Across from each single strand of DNA, we typically see adenine pair with thymine , and cytosine pair with guanine to form a parallel complementary strand as described below. Two nucleotide sequences which correspond to each other in this manner are referred to as complementary: A frayed end refers to

1638-544: The same mouse and in mice of different age. Efficiency was higher in the skin, lung and kidney fibroblasts, and lower in heart fibroblasts and brain astrocytes. Furthermore, NHEJ efficiency declined with age. The decline was 1.8 to 3.8-fold, depending on the tissue, in the 5-month-old compared to the 24-month-old mice. Reduced capability for NHEJ can lead to an increase in the number of unrepaired or faultily repaired DNA double-strand breaks that may then contribute to aging. (Also see DNA damage theory of aging .) An analysis of

1680-403: The two DNA strands four base pairs from each other, creating a four-base 3' overhang in one molecule and a complementary 3' overhang in the other. These ends are called cohesive since they are easily joined back together by a ligase. For example, these two "sticky" ends (four-base 5' overhangs) are compatible: Also, since different restriction endonucleases usually create different overhangs, it

1722-624: The two proteins can form hetero-oligomers via head-to-head interaction of alternating XLF and XRCC4 subunits. These XRCC4-XLF filaments have been proposed to bridge DNA prior to end ligation during NHEJ . Formation of XRCC4-XLF oligomers can be disrupted by interaction of the C-terminal domain of XRCC4 with the BRCT domain of DNA ligase IV. Deficiency of NHEJ1 in mice leads to premature aging of hematopoietic stem cells as indicated by several lines of evidence including evidence that long-term repopulation

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1764-442: The two strands run in opposite directions. Therefore, one end of the molecule will have the 3' end of strand 1 and the 5' end of strand 2, and vice versa in the other end. However, the fact that the molecule is two stranded allows numerous different variations. The simplest DNA end of a double stranded molecule is called a blunt end . Blunt ends are also known as non-cohesive ends. In a blunt-ended molecule, both strands terminate in

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