page_title: DNA repair
section_title: Double-strand breaks
hierarchical_section_title: DNA repair / Mechanisms / Double-strand breaks
caption_attribution_description: English: (Left panel): Canonical C-NHEJ. The heterodimer Ku80-Ku70 binds to DNA ends, which then recruits DNA-PKcs. In subsequent steps, several proteins including Artemis, polynucleotide kinase (PNK), and members of the polymerase X family process the DNA ends. In the last step, ligase IV associated with its co-factors Xrcc4 and Cernunos/XLF joins the ends. (Right Panel): Resection as a common initiation step for HR and A-EJ at DSB. 53BP1, RIF1 and Ku70-80 heterodimer protect DSB ends from resection and HR and A-EJ actions. The CDK1/2-dependent phosphorylation of CtIP and EXO1 favors the initiation of resection and extension, respectively. Recently, REV7/MAD2L2 was described as an inhibitor of resection and HR, although its role in A-EJ inhibition was not directly studied and remains hypothetical. A short ssDNA resection allows for A-EJ but not homologous recombination, while a long ssDNA resection allows for both A-EJ and HR; however, HR requires the presence of homologous sequences. Recently, POLQ polymerase was shown to inhibit HR and to promote A-EJ at DSBs. A-EJ results in repair that is error-prone and is associated with deletions at the repair junctions with frequent use of microhomologies that are distant from the DSB. Alternative-EJ: Parp1 plays a role in the initiation process, and it has been proposed that a single-strand DNA resection reveals complementary microhomologies (two to four nucleotides or more in length) that can anneal, with gap-filling completing the end-joining. A-EJ is always associated with deletions at the junctions and can involve microhomologies (MMEJ or microhomologies-mediated EJ) that are distant from the DSB. Subsequently, Xrcc1 and ligase III (which can be substituted by ligase I) complete the A-EJ process. Homologous recombination: The first step, which is the initiation of resection, involves the removal of ~50–100 bases of DNA from the 5' end by the MRN complex (Mre11-Rad50-Nbs1) in conjunction with CtIP. The second step, resection extension, is carried out by two alternate pathways involving either the 5' to 3' exonuclease EXO1 or the helicase-topoisomerase complex BLM-TOPIIIα-RMI1-2 in concert with the nuclease CtIP/DNA2. WRN helicase has also been shown to act with CtIP and to stimulate resection in human cells.&#91;1&#93;
context_page_description: DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA damage, resulting in as many as 1 million individual molecular lesions per cell per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. When normal repair processes fail, and when cellular apoptosis does not occur, irreparable DNA damage may occur, including double-strand breaks and DNA crosslinkages. This can eventually lead to malignant tumors, or cancer as per the two hit hypothesis.
  The rate of DNA repair is dependent on many factors, including the cell type, the age of the cell, and the extracellular environment.
context_section_description: Double-strand breaks, in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements. In fact, when a double-strand break is accompanied by a cross-linkage joining the two strands at the same point, neither strand can be used as a template for the repair mechanisms, so that the cell will not be able to complete mitosis when it next divides, and will either die or, in rare cases, undergo a mutation. Three mechanisms exist to repair double-strand breaks (DSBs): non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination (HR). In an in vitro system, MMEJ occurred in mammalian cells at the levels of 10–20% of HR when both HR and NHEJ mechanisms were also available.
  In NHEJ, DNA Ligase IV, a specialized DNA ligase that forms a complex with the cofactor XRCC4, directly joins the two ends. To guide accurate repair, NHEJ relies on short homologous sequences called microhomologies present on the single-stranded tails of the DNA ends to be joined. If these overhangs are compatible, repair is usually accurate. NHEJ can also introduce mutations during repair. Loss of damaged nucleotides at the break site can lead to deletions, and joining of nonmatching termini forms insertions or translocations. NHEJ is especially important before the cell has replicated its DNA, since there is no template available for repair by homologous recombination. There are "backup" NHEJ pathways in higher eukaryotes. Besides its role as a genome caretaker, NHEJ is required for joining hairpin-capped double-strand breaks induced during V(D)J recombination, the process that generates diversity in B-cell and T-cell receptors in the vertebrate immune system.
  Homologous recombination requires the presence of an identical or nearly identical sequence to be used as a template for repair of the break. The enzymatic machinery responsible for this repair process is nearly identical to the machinery responsible for chromosomal crossover during meiosis. This pathway allows a damaged chromosome to be repaired using a sister chromatid (available in G2 after DNA replication) or a homologous chromosome as a template. DSBs caused by the replication machinery attempting to synthesize across a single-strand break or unrepaired lesion cause collapse of the replication fork and are typically repaired by recombination.
  MMEJ starts with short-range end resection by MRE11 nuclease on either side of a double-strand break to reveal microhomology regions.  In further steps, Poly (ADP-ribose) polymerase 1 (PARP1) is required and may be an early step in MMEJ.  There is pairing of microhomology regions followed by recruitment of flap structure-specific endonuclease 1 (FEN1) to remove overhanging flaps.  This is followed by recruitment of XRCC1–LIG3 to the site for ligating the DNA ends, leading to an intact DNA. MMEJ is always accompanied by a deletion, so that MMEJ is a mutagenic pathway for DNA repair.
  The extremophile Deinococcus radiodurans has a remarkable ability to survive DNA damage from ionizing radiation and other sources. At least two copies of the genome, with random DNA breaks, can form DNA fragments through annealing. Partially overlapping fragments are then used for synthesis of homologous regions through a moving D-loop that can continue extension until they find complementary partner strands. In the final step there is crossover by means of RecA-dependent homologous recombination.
  Topoisomerases introduce both single- and double-strand breaks in the course of changing the DNA's state of supercoiling, which is especially common in regions near an open replication fork. Such breaks are not considered DNA damage because they are a natural intermediate in the topoisomerase biochemical mechanism and are immediately repaired by the enzymes that created them.
caption: Double-strand break repair pathway models
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