DNA repair

Parent
DNA metabolism
Subordinate
Single- strand break repair Double-strand break repair Post-replication repair Viral DNA repair Mitochondrial DNA repair Pyrimidine dimer repair Base excision repair Nucleotide excision repair
Gene Ontology
QuickGO

Through mechanisms of DNA repair cells can eliminate changes in their DNA structure. Such damage to the DNA can be caused spontaneously in the course of DNA replication or by exposure to mutagenic substances, extreme heat or ionizing radiation .

DNA damage can lead to the incorrect replication of the DNA for mitosis , proteins no longer or incorrectly synthesized or important chromosome areas being split off after double-strand breaks .

If the complex repair mechanisms of the cell are unsuccessful, so many defects accumulate in growing and resting somatic cells that normal cell functions are disturbed. In a germ cell, the daughter cells would no longer be viable, which leads to an inactivation of the cell line: the cell or the second to third subsequent generations lose their ability to divide and die. In the course of cell cycle control , control proteins can recognize a cell or its DNA as defective and initiate cycle arrest (G ₀ phase) or programmed cell death ( apoptosis ).

In the meantime, it has been possible to follow individual DNA repair enzymes while working in a bacterium with PAL microscopy and to determine the corresponding parameters. In E. coli, for example, a base excision repair takes a good two seconds.

Causes of DNA Damage

Possible causes are metabolic processes, chemical substances or ionizing radiation such as UV radiation , electrons or protons .

DNA damage as well as errors in replication and other cellular processes can lead to mutations . Since mutations have their own repair mechanisms, they can be viewed here in a similar way to DNA damage.

Metabolic processes

A cell is a steady state system . It continually absorbs molecules, processes them, synthesizes the substances it needs, and in turn releases certain substances into the environment. During normal cellular metabolism , reactive oxygen species (ROS, including oxygen radicals) can arise, which cause a significant amount of oxidative damage. Most often these are base damage and single-strand breaks, less than 0.5% are double-strand breaks, which are also distributed relatively uniformly over the DNA. The probability of endogenously induced clusters of damage and thus - difficult to repair - accumulated lesions (complex lesions), as they otherwise occur due to the non-homogeneous release of energy from ionizing radiation, is very low. Too high a proton density and / or too high a temperature can trigger depurination or depyrimidation .

UV radiation

The UV radiation can lead to direct changes ( mutations ) in the DNA, which in particular absorbs UV-C-FUV radiation. Single-stranded DNA shows its absorption maximum at 260 nm. Both UV-B and UV-A can indirectly damage the DNA through the formation of reactive oxygen radicals , which cause the formation of oxidative DNA lesions, which in turn lead to mutations. These are presumably responsible for the development of UV-A-induced tumors.

Types of DNA Damage

→ Main article : DNA damage

• Base modifications
  • Pyrimidine dimers usually 6–4 photo products ( 6-4PPs ) or cyclobutane pyrimidine dimers ( CPDs )
  • oxidized bases for example 8-oxo-7,8-dihydroguanine ( 8-oxoG ) or 8-oxo-7,8-dihydroadenine ( 8-oxoA )
  • alkylated bases (e.g. base methylation)
  • other Bulky lesions (bulky base changes)
• Base mismatches due to incorrect replication ( mismatch )
• Loss of base - apurination or apyrimidation ( AP sites )
• Changes in the sugar structure
• DNA-protein crosslinks ( DNA-protein crosslinks )
• DNA-DNA links ( DNA crosslinks )
• Single strand breaks ( ss breaks )
• Double strand breaks ( ds breaks )

The treatment with a Gray X-ray radiation generates approximately per cell

• 1000-2000 base modifications
• 500–1000 single strand breaks
• 800–1600 Changes in the sugar structure
• 150 DNA-protein cross-links
• 50 double strand breaks

Repair of DNA damage

DNA repair

Different specialized repair mechanisms exist for the different types of DNA damage. For example, some mechanisms specialize in repairing damage to single DNA strands, while others specialize in repairing DNA double-strand breaks.

There are also differences between prokaryotes and eukaryotes, which have different DNA polymerases .

Single strand damage repair

Base Excision Repair (BER)

Base excision repair fixes defects in the form of oxidized, alkylated, or deaminated individual bases. Damage to the bases is recognized by a specific DNA glycosylase and cut out (excision). This migrates along the small groove and folds the individual bases into their catalytic center. A damaged base is removed from the DNA glycosylase, after which an AP endonuclease (apurinic / apyrimidinic endonuclease) introduces a single-strand break in the sugar-phosphate backbone. A DNA polymerase synthesizes the correct base depending on the complementary base on the fault-free strand. There are two variants of BER: short patch repair (a single base is replaced) and long patch repair (2-20 nucleotides are replaced). In humans, the DNA polymerase β ( Pol β ) is the main polymerase responsible. A DNA ligase links the new base in the DNA strand, which corrects the error.

Nucleotide Excision Repair (NER)

In contrast to base excision repair, the nucleotide excision repair (NER) primarily detects so-called “bulky lesions”, i.e. places that create a kind of “hump” in the DNA molecule and thereby disrupt the helical structure. These can be pyrimidine dimers and 6.4 photo products which are generated by UV radiation.

Nucleotide excision repair is divided into damage detection, incision, cutting out a 25-30 base long DNA segment, the new synthesis of this segment and the subsequent ligation.

NER occurs in both prokaryotes and eukaryotes, but the mechanisms and enzymes involved differ. While in prokaryotes like Escherichia coli Uvr proteins and DNA polymerase I are involved, in eukaryotes it is proteins that get their names from the hereditary diseases Xeroderma pigmentosum and Cockayne syndrome , e.g. B. XPA or CSA . The polymerases involved include the DNA polymerases δ, ε and / or κ.

In eukaryotes, there are two routes to nucleotide excision repair. On the one hand, Global Genome Repair (GGR), which repairs damage in transcription-inactive areas of the DNA, and on the other hand, the so-called Transcription Coupled Repair (TCR), which repairs damage to the DNA currently to be transcribed. The only difference between these two forms is the detection of damage.

In TCR, it is important that the RNA polymerase II blocked by the damage is removed so that the TCR proteins can access the DNA damage. This removal of RNA polymerase II is made possible by CSA and CSB. In GGR, the DNA lesion is recognized by the protein complex XPC / HHR23B. In contrast, this complex plays no role in TCR. The further steps are identical for both repair methods. XPA and RPA are used for further DNA damage detection and they direct the helicases XPB and XPD to the lesion, which unwind the DNA in the immediate vicinity of the damage.

In the last few years the fate of the moonlight children has been increasingly pointed out in the media. These are children who are affected by a rare genetic defect and who must avoid all exposure to sunlight in order to counteract the rapid development of skin cancer. These children suffer from xeroderma pigmentosum (XP, also called melanosis lenticularis progressiva or moonshine disease), which is caused by a defect in the repair of nucleotides. The defect not only results in xeroderma pigmentosum, but also two other diseases, the so-called Cockayne syndrome and trichothiodystrophy.

The endonucleases XPG and XPF-ERCC1 cut the DNA strand at two points, 3 'and 5' (dual incision), so that an oligonucleotide of about 30 bases is released which contains the damage. This is followed by the polymerisation of the missing DNA segment by DNA polymerase and other factors. The last step is the ligation of the synthesized section by DNA ligase I and flap endonuclease 1 or the ligase III-XRCC1 complex.

Mutations affecting the XPA-XPG family lead to the development of the clinical picture Xeroderma pigmentosum . With Xeroderma pigmentosum the risk of skin cancer is increased, which indicates the importance of a functioning DNA repair after UV radiation.

Proofreading by DNA polymerase (base mismatch repair, mismatch repair)

The protein DNA polymerase, which is responsible for copying the DNA, is able to check the new DNA strand during synthesis and compare it with the original strand. However, this function is imprecise and without further control by DNA mismatch repair proteins , the number of spontaneous mutations would be 1000-fold increased. The bacterium Escherichia coli can also use its methylation status to differentiate the faulty daughter strand that has arisen during replication from the copied DNA. The new strand is methylated somewhat later than the template (the parent strand) on the adenine residues of the GATC sequence . A defect in the Mismatchreparatur causes a form of colon cancer: hereditary nonpolyposis colorectal cancer (HNPCC, hereditary nonpolyposis colorectal cancer ).

Photoreactivation

Photolyases are able to dissolve cyclobutane rings and (6-4) photo products formed in the DNA by ultraviolet radiation . They have a so-called antenna complex with which they absorb blue or ultraviolet light and, with the help of this energy, transfer two electrons from the cofactor FAD to the DNA damage bound in the active center of the enzyme . The same split as a result. The photolyase thus restores the native structure of the DNA without cutting out or inserting bases . To date, photolyases have been detected in many organisms from prokaryotes to fungi and plants to marsupials . Despite their undisputed beneficial properties for these organisms, the photolyases have been lost several times in the course of evolution. The higher mammals , to which humans belong, no longer have any repair-active variants of these proteins. The reasons for this have not yet been conclusively clarified.

Repair of double strand breaks

A double-strand break can generally be repaired using homologous repair mechanisms or non-homologous repair. Non-homologous repair is more prone to errors and more often leads to changes in the original sequence in the form of deletions or smaller insertions. This property is made use of in genome editing, for example through the CRISPR / Cas9 system, in order to generate targeted mutations in a genome. Homologous repair mechanisms are particularly common in bacteria and yeasts, while in higher eukaryotes most double-strand breaks are repaired non-homologously. Homologous repair mechanisms are particularly active during the S and G2 cell phase. The sister chromatid is then often near the break point and can serve as a template for the repair. In the G1 to early S phase, non-homologous repair is more active.

Homologous repair

Homologous repair begins with resection of the 5 'ends of an open hernia by the MRX complex (MRN in mammals and plants) consisting of MRE11, Rad50 and XRCC1 (NBS1). With the help of the proteins Rad51, Rad54, among other things, the single-stranded 3 'end of the break binds in a region homologous to the repair site (e.g. the sister chromatid) and forms a structure called a D-loop . The missing strand is synthesized using the homologous template. Two Holliday structures are then formed, the dissolution of which either results in a crossover of the two strands or in a dissolution.

Synthesis dependent strand annealing (SDSA)

If the binding of the open end of the double-strand break takes place in an area that is not homologous to the repair site, it is called SDSA. As a result, the break is repaired, but so-called filler sequences are incorporated that come from the area where the D-loop was formed.

Single strand annealing

If there is a break within between repeats, the ends of the break can be shortened to such an extent that homologous pairing can take place in longer areas of homology. The strand is then repaired, which leads to a deletion of the area between the two repeats.

Non-homologous repair

Non-homologous repair is done without using a homologous template. A distinction must be made between non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ) - sometimes also called alternative or backup NHEJ.

Non-homologous end-joining (NHEJ)

NHEJ starts by binding the Ku complex consisting of Ku70 and Ku80 to the open ends of the double-strand break. This protects the open ends from further degradation by exonucleases and the break is stabilized. When binding the open ends, short areas of homology between the open ends (so-called microhomology) are often used. Then the binding of the MRX or MRN complex takes place (see homologous repair), which processes the open ends so that they can be repaired by the XRCC4-DNA-LigaseIV complex. NHEJ is often described as a failure-prone repair mechanism. Repair by NHEJ usually results in smaller deletions of a few bases or small insertions at the repair site.

Microhomology-mediated end-joining (MMEJ)

If the Ku complex does not bind to an open end, e.g. in knockout mutants or during the S or G2 phase, when Ku70 / 80 is not as active, the stabilization of the open ends takes place in longer areas of microhomology (5- 25 bp). The open break is then processed by the MRX / MRN complex and repaired by XRCC4 / DNA ligase IV. Without the protection of the Ku complex, the open ends are exposed to exonucleases for longer. This and the use of a larger area of ​​microhomology at the repair site can result in larger deletions.

A disruption of these repair systems often manifests itself clinically as chromosome breakage syndrome , such as Nijmegen breakage syndrome .

Repair of cross-links

Cross -linked DNA leads to an activation of the proteins RAD18-SLF1-SLF2 in vertebrate cells, whereby the cross-linked DNA is ubiquitinated by RNF8 / RNF168 and then the SMC5 / 6 protein complex binds.

Individual evidence

1. ↑ CR Bartram: Genetic bases of carcinogenesis. In: W. Hiddemann, CR Bartram (Ed.): Die Onkologie. Part 1, Edition 2, Verlag Springer, 2009, ISBN 3-540-79724-6 , pp. 118–127 ( limited preview in Google Book Search).
2. ↑ S. Uphoff, R. Reyes-Lamothe u. a .: Single-molecule DNA repair in live bacteria. In: Proceedings of the National Academy of Sciences . Volume 110, number 20, May 2013, pp. 8063-8068, doi: 10.1073 / pnas.1301804110 . PMID 23630273 . PMC 3657774 (free full text).
3. ↑ Sung-Lim Yu, Sung-Keun Lee: Ultraviolet radiation: DNA damage, repair, and human disorders. In: Molecular & Cellular Toxicology . 13, 2017, p. 21, doi : 10.1007 / s13273-017-0002-0 .
4. ↑ Peter Elsner, Erhard Hoelzle a. a .: Daily sun protection in the prevention of chronic UV damage to the skin. In: JDDG. 5, 2007, doi : 10.1111 / j.1610-0387.2007.06099_supp.x .
5. ^ Rolf Sauer : Radiation Therapy and Oncology. 5th edition, Elsevier GmbH, Urban and Fischer Verlag, Munich, 2010; ISBN 978-3-437-47501-6 , p. 112 ( limited preview in Google book search).
6. ↑ Lucas-Lledó, JI & Lynch, M. Evolution of mutation rates: phylogenomic analysis of the photolyase / cryptochrome family . Mol. Biol. Evol 26: 1143-1153 (2009).
7. ↑ M. Raschle, G. Smeenk, RK Hansen, T. Temu, Y. Oka, MY Hein, N. Nagaraj, DT Long, JC Walter, K. Hofmann, Z. Storchova, J. Cox, S. Bekker-Jensen , N. Milan, M. Mann: Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links. In: Science. 348, 2015, p. 1253671, doi: 10.1126 / science.1253671 .