What is nucleotide excision repair

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Fei, J. PLoS Biol. Fischer, E. Cell , — Fitch, M. Fong, Y. Gale, J. UV-induced formation of pyrimidine dimers in nucleosome core DNA is strongly modulated with a period of UV-induced photoprducts are distributed differently than cyclobutane dimers in nucleosomes. Garinis, G.

Transcriptome analysis reveals cyclobutane pyrimidine dimers as a major source of UV-induced DNA breaks. Groisman, R. Guerrero-Santoro, J. Cancer Res. Hanawalt, P. Transcription-coupled DNA repair: two decades of progress and surprises. Structure 19, — He, J. Hey, T. Biochemistry 41, — Hollander, M. Deletion of XPC leads to lung tumors in mice and is associated with early events in human lung carcinogenesis. Huang, J. Hwang, B.

Expression of the p48 xeroderma pigmentosum gene is pdependent and is involved in global genomic repair. Itoh, T. DDB2 gene disruption leads to skin tumors and resistance to apoptosis after exposure to ultraviolet light but not a chemical carcinogen. Jing, Y. Thermodynamic and base-pairing studies of matched and mismatched DNA dodecamer duplexes containing cis-syn, and Dewar photoproducts of TT.

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Biochemistry Moscow 77, — Kulaksiz, G. Kuraoka, I. Li, C. Liu, L. Lopes, M. Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions. Lubin, A. A human XPC protein interactome—a resource. Marteijn, J. Understanding nucleotide excision repair and its roles in cancer and ageing. Masutani, C. Since this transition is not repaired, over time the number of CpG dinucleotides is greatly diminished in the genomes of vertebrates and plants.

Methyl- Replicate. Methylation of CpG dinucleotides followed by oxidative deamination results in TpG dinucleotides. Some regions of plant and vertebrate genomes do not show the usual depletion of CpG dinucleotides. Instead, the frequency of CpG approaches that of GpC or the frequency expected from the individual frequency of G and C in the genome.

They are distinctive regions of these genomes and are often found in promoters and other regulatory regions of genes. Examination of several of these CpG islands has shown that they are not methylated in any tissue, unlike most of the other CpGs in the genome.

Current areas of research include investigating how the CpG islands escape methylation and their role in regulation of gene expression. If a CpG island were to be methylated in the germ line, what would be consequences be over many generations?

The rate of oxidation of bases in DNA can be increased by treating with appropriate reagents, such as nitrous acid HNO 2. Many mutagens are alkylating agents. This means that they will add an alkyl group, such as methyl or ethyl, to a base in DNA. The chemical warfare agents sulfur mustard and nitrogen mustard are also alkylating agents. N-methyl-nitrosoguanidine and MNNG transfer a methyl group to guanine e. The additional methyl or other alkyl group causes a distortion in the helix.

The distorted helix can alter the base pairing properties. For instance, O 6 -methylguanine will sometimes base pair with thymine Fig. The order of reactivity of nucleophilic centers in purines follows roughly this series:. The products of this reaction are primarily N 7 -guanine, but N 3 -adenine is also detectable. This reaction is used to identify protein-binding sites in DNA, since interaction with a protein can cause decreased reactivity to DMS of guanines within the binding site but enhanced reactivity adjacent to the site.

Methylation to form N 7 -methyl-guanine does not cause miscoding in the DNA, since this modified purine still pairs with C. Some compounds cause a loss of nucleotides from DNA. If these deletions occur in a protein-coding region of the genomic DNA, and are not an integral multiple of 3, they result in a frameshift mutation.

These are commonly more severe loss-of-function mutations than are simple substitutions. Frameshift mutagens such as proflavin or ethidium bromide have flat, polycyclic ring structures Fig. They may bind to and intercalate within the DNA, i. If a segment of the template DNA is the looped out, DNA polymerase can replicate past it, thereby generating a deletion.

Intercalating agents can stabilize secondary structures in the loop, thereby increasing the chance that this segment stays in the loop and is not copied during replication Fig.

Thus growth of cells in the presence of such intercalating agents increase the probability of generating a deletion. Two intercalating agents A and their ability to stabilize loops in the template, leading to deletions in the nascent DNA strand B. Benz a pyrenes are present in soot. High energy radiation, such as X-rays, g -rays, and b particles or electrons are powerful mutagens. Since they can change the number of electrons on an atom, converting a compound to an ionized form, they are referred to as ionizing radiation.

They can cause a number of chemical changes in DNA, including directly break phosphodiester backbone of DNA, leading to deletions. Ionizing radiation can also break open the imidazole ring of purines. Subsequent removal of the damaged purine from DNA by a glycosylase generates an apurinic site.

Ultraviolet radiation with a wavelength of nm will form pyrimidine dimers between adjacent pyrimidines in the DNA. The dimers can be one of two types Fig. The major product is a cytobutane-containing thymine dimer between C5 and C6 of adjacent T's. The other product has a covalent bond between position 6 on one pyrimidine and position 4 on the adjacent pyrimidine, hence it is called the "" photoproduct. Pyrimidine dimers formed by UV radiation, illustrated for adjacent thymidylates on one strand of the DNA.

A Formation of a covalent bond between the C atoms at position 5 of each pyrimidine and between the C atoms at position 6 of each pyrimidine makes a cyclobutane ring connecting the two pyrimidines. The bases are stacked over each other, held in place by the cyclobutane ring. The C-C bonds between the pyrimidines are exaggerated in this drawing so that the pyrimidine ring is visible. B Another photoproduct is made by forming a bond between the C atom at position 6 of one pyrimidine and position 4 of the adjacent pyrimidine, with loss of the O previously attached at position 4.

The pyrimidine dimers cause a distortion in the DNA double helix. This distortion blocks replication and transcription. What is the physical basis for this distortion in the DNA double helix?

Table 7. Note that some of these mutations lead to mispairing substitutions , others lead to distortions of the helix, and some lead to both. Transitions can be generated both by damage to the DNA and by misincorporation during replication. Transversions occur primarily by misincorporation during replication. The frequency of such errors is greatly increased in mutator strains, e. Also, after a bacterial cell has sustained sufficient damage to induce the SOS response, the DNA polymerase shifts into a an error-prone mode of replication.

This can also be a source of mutant alleles. Summary of effects of various agents that alter DNA sequences mutagens and mutator genes.

Repair mechanisms. The second part of this chapter examines the major classes of DNA repair processes. These are:. Many of these processes were first studies in bacteria such as E. For instance, nucleotide excision repair and base excision repair are found in virtually all organisms, and they have been well characterized in bacteria, yeast, and mammals.

Like DNA replication itself, repair of damage and misincorporation is a very old process. Some kinds of covalent alteration to bases in DNA can be directly reversed. This occurs by specific enzyme systems recognizing the altered base and breaking bonds to remove the adduct or change the base back to its normal structure. Photoreactivation is a light-dependent process used by bacteria to reverse pyrimidine dimers formed by UV radiation.

The enzyme photolyase binds to a pyrimidine dimer and catalyzes a second photochemical reaction this time using visible light that breaks the cyclobutane ring and reforms the two adjacent thymidylates in DNA. Note that this is not formally the reverse of the reaction that formed the pyrimidine dimers, since energy from visible light is used to break the bonds between the pyrimidines, and no UV radiation is released.

The photolyase enzyme has two subunits, which are encoded by the phrA and phrB genes in E. A second example of the reversal of damage is the removal of methyl groups.

It then removes the methyl group, transferring it to an amino acid of the enzyme. The methylated enzyme is no longer active, hence this has been referred to as a suicide mechanism for the enzyme. The most common means of repairing damage or a mismatch is to cut it out of the duplex DNA and recopy the remaining complementary strand of DNA, as outlined in Fig. Three different types of excision repair have been characterized: nucleotide excision repair, base excision repair, and mismatch repair.

All utilize a cut, copy, and paste mechanism. In the cutting stage, an enzyme or complex removes a damaged base or a string of nucleotides from the DNA. The DNA polymerase can initiate synthesis from 3' OH at t he single-strand break nick or gap in the DNA remaining at the site of damage after excision. A general scheme for excision repair, illustrating the cut steps 1 and 2 , copy step 3 and paste step 4 mechanism.

In nucleotide excision repair NER , damaged bases are cut out within a string of nucleotides, and replaced with DNA as directed by the undamaged template strand. This repair system is used to remove pyrimidine dimers formed by UV radiation as well as nucleotides modified by bulky chemical adducts. The common feature of damage that is repaired by nucleotide excision is that the modified nucleotides cause a significant distortion in the DNA helix. NER occurs in almost all organisms examined.

The genes encoding this repair function were discovered as mutants that are highly sensitive to UV damage, indicating that the mutants are defective in UV repair. As illustrated in Fig. Mutant strains can be identified that are substantially more sensitive to UV radiation; these are defective in the functions needed for UV - r esistance, abbreviated uvr. By collecting large numbers of such mutants and testing them for their ability to restore resistance to UV radiation in combination, complementation groups were identified.

Survival curve of bacteria exposed to UV radiation. Cultures of bacteria are exposed to increasing doses of UV radiation, plotted along the horizontal axis. Samples of each irradiated culture are then plated and the number of surviving colonies are counted plotted as a logarithmic function on the vertical axis. Mutant strains that are more sensitive to UV damage are defective in the genes that confer UV - r esistance, i.

The enzymes encoded by the uvr genes have been studied in detail. It then cleaves on both sides of the damage. Thus for this system, the UvrABC and UvrD proteins carry out a series of steps in the cutting phase of excision repair.

The UvrABC proteins form a dynamic complex that recognizes damage and makes endonucleolytic cuts on both sides. The two cuts around the damage allow the single-stranded segment containing the damage to be excised by the helicase activity of UvrD. After the damaged segment has been excised, a gap of 12 to 13 nucleotides remains in the DNA. In more detail, the process goes as follows Fig.

UvrA 2 then dissociates, in a step that requires ATP hydrolysis. The UvrBC complex is the active nuclease. It makes the incisions on each side of the damage, in another step that requires ATP. The phosphodiester backbone is cleaved 8 nucleotides to the 5' side of the damage and nucleotides on the 3' side. Like all helicase reactions, the unwinding requires ATP hydrolysis to disrupt the base pairs.

Thus ATP hydrolysis is required at three steps of this series of reactions. The nucleotide-long fragment is released together with the excinuclease by helicase II action.

How does an excinuclease differ from an exonuclease and an endonuclease? Nucleotide excision repair is very active in mammalian cells, as well as cells from may other organisms.

The DNA of a normal skin cell exposed to sunlight would accumulate thousands of dimers per day if this repair process did not remove them!

One human genetic disease, called xeroderma pigmentosum XP , is a skin disease caused by defect in enzymes that remove UV lesions. Fibroblasts isolated from individual XP patients are markedly sensitive to UV radiation when grown in culture, similar to the phenotype shown by E.

These XP cell lines can be fused in culture and tested for the ability to restore resistance to UV damage. XP cells lines that do so fall into different complementation groups. Several complementation groups, or genes, have been defined in this way. Considerable progress has been made recently in identifying the proteins encoded by each XP gene Table 7.

Note the tight analogy to bacterial functions needed for NER. Similar functions are also found in yeast Table 7. NER occurs in two modes in many organisms, including bacteria, yeast and mammals. One is the global repair that acts throughout the genome, and the second is a specialized activity is that is coupled to transcription. In transcription coupled NER, the elongating RNA polymerase stalls at a lesion on the template strand; perhaps this is the damage recognition activity for this mode of NER.

A rare genetic disorder in humans, Cockayne syndrome CS , is associated with a defect specific to transcription coupled repair. Determination of the nature and activity of the proteins encoded by them will provide additional insight into the efficient repair of transcribed DNA strands. The phenotype of CS patients is pleiotropic, showing both photosensitivity and severe neurological and other developmental disorders, including premature aging.

These symptoms are more severe than those seen for XP patients with no detectable NER, indicating that transcription-coupled repair or the CS proteins have functions in addition to those for NER. Other genetic diseases also result from a deficiency in a DNA repair function, such as Bloom's syndrome and Fanconi's anemia. These are intensive areas of current research.

Ataxia telangiectasia, or AT, illustrates the effect of alterations in a protein not directly involved in repair, but perhaps signaling that is necessary for proper repair of DNA.

AT is a recessive, rare genetic disease marked by uneven gait ataxia , dilation of blood vessels telangiectasia in the eyes and face, cerebellar degeneration, progressive mental retardation, immune deficiencies, premature aging and about a fold increase in susceptibility to cancers.

The gene that is m utated in AT hence called "ATM" was isolated in and localized to chromosome 11q In addition, genomic DNA is organized into chromatin structures, which are quite heterogeneous and sometimes highly condensed, thereby preventing access to DNA binding protein factors.

Therefore, one can reasonably assume existence of specialized molecular mechanisms, which assist XPC to discriminate its rare target sites efficiently from intact DNA present in large excess. Its binding to pyrimidine 6—4 pyrimidone photoproducts 6—4PPs is especially strong, whereas CPDs show moderate but significant affinity [ 27 , 28 ]. In contrast, bulky base adducts induced by chemical mutagens are relatively poor substrates [ 29 , 30 ]. Biochemical studies have shown that DNA lesions within the nucleosome core tend to refrain from interaction with XPC and the following repair reaction [ 37 , 38 ].

Because 6—4PPs are shown to be generated by UV irradiation in chromatin in a relatively random manner [ 46 ], this fact strongly suggests existence of unprecedented molecular mechanisms that enable alteration of chromatin structures prior to lesion detection by XPC. We have recently reported that XPC physically interacts with histone H3 and this interaction is negatively regulated by acetylation of the histone protein [ 47 ].

Based on these findings, we propose that DNA damage associated with a relatively large helix distortion may be able to induce local reorganization of chromatin including deacetylation of histones, which may contribute to efficient recruitment of XPC [ 47 ]. Since chemical changes in DNA caused by lesions per se are only small, roles for chromatin structures could be completely different from the situation of UV-DDB-bound lesions.

In order to explore novel molecular mechanisms underlying regulation of DNA damage recognition for GG-NER, we have recently set up a confocal laser scanning microscopy equipped with a nm femtosecond fiber laser, with which local DNA damage similar to that induced by nm UV irradiation can be generated within cell nuclei by a principle of three-photon absorption.

This system enables us to observe retarded accumulation of fluorescence-tagged XPC to the damaged sites after suppression of DDB2 expression or treatment with a histone deacetylase inhibitor Fig. DNA damage was induced with the nm femtosecond laser and three-photon absorption within subnuclear regions of human osteosarcoma U2OS cells stably expressing mCherry-fused XPC. Mean values and standard deviations were calculated from 10 a and 17 b samples, respectively.

Utilizing intrinsic sites that allow XPC binding, it is possible that this stepwise strategy further extends potential of lesion recognition in GG-NER. One of our goals would be to recapitulate the GG-NER processes with damaged chromatin substrates in the cell-free system.

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