Which Of The Following Statements About Repair Of Thymine Dimers Is True?

Enzymatic recognition of radiation-produced oxidative Deoxyribonucleic acid lesion. Molecular dynamics arroyo

Miroslav Pinak , in Modern Methods for Theoretical Physical Chemistry of Biopolymers, 2006

x.5.2 Thymine dimer (five,6 cis.sin cyclobuthane thymine dimer)

Cyclobuthane thymine dimer is a photolesion produced by UV radiation in sunlight and is considered as a potential factor causing pare cancer. Information technology is formed as a covalently bonded complex of 2 adjacent thymines on a unmarried strand of DNA. This damage is very frequent but nearly xc pct of TDs are repaired within a short time of the order of minutes, and merely a few are experimentally observable and originate future changes at the jail cell level.

This study was conducted with DNA dodecamer d(TCGCG′TD′GCGCT)₂, where TD refers to thymine dimer [10]. The results of 600 ps of Medico simulation signal that this lesion does not disrupt the double helical construction and the hydrogen bonds are well preserved throughout the simulation. Instead, a thymine dimer lesioned DNA, if compared with a non-lesioned one, has sharp angle at the dimer site which is originated by two covalent bonds C(five)-C(5) and C(half-dozen)-C(6) between next thymines forming thymine dimer (Fig. 10.ii). This bending is supposed to discriminate lesion from undamaged DNA segment and to originate conformation that facilitates the formation of a Deoxyribonucleic acid–enzyme circuitous past generating complementary structural shapes of repair enzymes and bent DNA.

Thymine dimer excision repair is initiated past E. coli endonuclease V of bacteriophage T4 that slides on non-target sequences and progressively incises at all dimers inside the Deoxyribonucleic acid molecule. This enzyme binds to the Deoxyribonucleic acid double strand in a two-step process: at showtime it scans not-target Dna by electrostatic interaction to search for damaged sites, and secondly it sequentially specifically recognizes the dimer sites. The process of binding of T4 endonuclease 5 to thymine dimer lesioned Dna was simulated using the Doc method. Because the limitations arising from the simulations of large systems and requirements for CPU fourth dimension, instead of the whole enzyme a small isolated part that included a catalytic center was subjected to the simulations. The office of the enzyme was selected considering the structure of T4 endonuclease V. This enzyme consists of three a helices (H1: amino acids 14–38, H2: 64–82 and H3: 108–124) standing side by side, several reverse turns and several loops [26]. Glu-23, of which a carboxyl chain plays a crucial function in the cleavage of the North-glycosyl bond in Deoxyribonucleic acid during enzymatic repair procedure [27, 28], is surrounded by amino acids Arg-three, Arg-22 and Arg-26 belonging to helix H1. The sidechain of Glu-23 also forms hydrogen bonds with the sidechains of helices H1 and H2, and lies on the molecular surface. Considering these properties, viii amino acids of H1, Glu-20, Tyr-21, Arg-22, Glu-23, Leu-24, Pro-25, Arg-26, Val-27, and two amino acids at the NH_ii terminus, Thr-2 and Arg-3, were selected to form the simulated function of the enzyme. Amino acids Thr-2, Arg-22, Glu-23 and Arg-26 course the catalytic centre that is active in the incision of the thymine dimmer during the repair procedure and together with other six selected amino acids: Arg-three, Glu-xx, Tyr-21, Leu-24, Pro-25 and Val-27, are located at the key function of a concave site of the enzyme and may be easily exposed to the DNA surface.

Doctor simulation showed that after nearly 100 ps of the Md simulation, the catalytic role of the enzyme approached the DNA at the thymine dimer site, docked into it, and this complex remained stable afterwards (the simulation was performed for 500 ps).

Examining the contact expanse between Dna and the enzyme it can exist observed that in that location is an intensive interaction arising from the shut proximity of Arg-22 and Tyr-21 to C3′, and Arg-26 to C5′ atoms of thymine dimer. In addition, Arg-26 comes very close to the C5′ atom. This specific position of Arg-26 determines the orientation of the enzyme toward the thymine dimer. It tin likewise exist noted that Arg-22 and Arg-26 contribute to the recognition and stability of the complex structure. Glu-23, cleaving the N-glycosyl bail in the repair procedure, is located close to the C5′ atom. These observations are in adept agreement with crystal structure, where side bondage of Arg-22 and Arg-26 form stacking contact with the Dna [29].

When the control simulation was performed with a not-lesioned Dna molecule, the selected part of the enzyme did not dock into the DNA molecule and the DNA–enzyme circuitous was not formed.

In this simulation only a relatively pocket-size part of the enzyme was used and thus the analysis of results is mainly focused on the description of the structural properties at the contact area and on the possible role of the electrostatic free energy in the recognition procedure, as will be discussed subsequently in the text.

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Computational Molecular Biology

Misako Aida , ... Michel Dupuis , in Theoretical and Computational Chemistry, 1999

4.4 Initial thymine dimer radical cation

The RHF adding on the thymine dimer in the previous section showed that the band fusion at the C5 and C6 atoms of 2 thymine bases created the four-member cyclobutane puckered ring. The aforementioned characteristic is observed with the CAS(3e+4o) level. The puckering leads to axial or equatorial directions for the substituent atoms on the cyclobutane ring: the substituent atoms on the ii thymine bases differ in their directionality. Especially noteworthy is the direction of the H6A atom. In the neutral T<>T, the H6A atom is equatorial relative to the four-member band and centric relative to the π conjugation system in the planar N1(H1)-C2(O2)-N3(H3)-C4(O4) moiety in ring A. Therefore, electrons on the C6A-H6A bond participate in the stabilization of the π system (i.due east., hyperconjugation). The highest occupied molecular orbital (HOMO) of the neutral T<>T is localized on the C6A-C6B bond.

Subsequently ionization, the SOMO orbital of TTp-1 is localized on the C6A-C6B bond (see Figure eight(a)), and the bond is lengthened (see Table viii) compared to the neutral species, every bit the bond is at present weaker due to the single electron occupancy. The iv-member ring in TTp-1 is puckered (come across Table 5), as in the neutral species. The relative orientations of the H6A atom in TTp-1 are similar to those in the neutral T<>T. Electrons on the C6A-H6A bond participate in the stabilization of the π system. The slightly higher spin population on the C6A cantlet than that on the C6B atom (run into Table vii) is probably caused by this result. The puckering of the cyclobutane ring is responsible for the departure in the electronic construction in the ii thymine rings and plays an of import role in determining the dissociation path as volition be described afterward.

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Enzymes and Enzyme Mechanisms

Eric G. Shepard , Joan B. Broderick , in Comprehensive Natural Products 2, 2010

8.17.iii.4 Deoxyribonucleic acid Repair: SPL

The major photoproduct in UV-irradiated Bacillus spore DNA is a unique thymine dimer called spore photoproduct (SP, 5-thyminyl-five,6-dihydrothymine). ^108–110 In contrast, UV irradiation of DNA in nearly growing cells produces primarily cyclobutane pyrimidine dimers as well as the half dozen,iv-photoproduct. ¹¹¹ The unusual UV photochemistry of Bacillus spores appears to be largely associated with the presence in spores of large quantities of a family of proteins known equally small-scale, acrid-soluble proteins (SASPs). ^112–115 It has been proposed that bounden of SASPs to DNA promotes a structural change and a modify in the level of hydration of the DNA that results in the formation of SP rather than cyclobutane thymine dimers. ^116–118 Pyrimidine dimers such as SP are damaging to cells, as they tin can block replication and transcription or can outcome in mutations if transcription proceeds past the region of the dimer. Repair of these dimers, therefore, is critical in order to avert mutations, and thus is the primal to UV resistance. Although pyrimidine dimers can be excised and replaced, the only well-characterized example of direct pyrimidine dimer reversal is the photoreactivation catalyzed by Deoxyribonucleic acid photolyase. ^111,119 Even so, photoreactivation has been shown to be absent in many species, including Bacillus, suggesting that alternating means of pyrimidine dimer repair might be plant. ^109,120

The enzyme SPL is the first identified nonphotoactivatable pyrimidine dimer lyase and it specifically targets SP and cleaves it into ii thymines by a light-independent mechanism. ^121,122 Early publications ^123,124 provided evidence that SPL utilized SAM and contained an iron–sulfur cluster, suggesting that it was a member of the Atomic number 26–S/AdoMet family of enzymes. Subsequent work has shown that the [4Fe–4S]⁺ state of SPL is active in SP repair and that SP repair is initiated past direct H-cantlet brainchild from the C6 of SP. ^125,126 Evidence that SP utilized SAM catalytically, and did not generate 5′-deoxyadenosine and methionine as products of turnover, placed SPL alongside LAM every bit the radical SAM enzyme that utilizes SAM every bit a cofactor. Post-obit on the initial reports of SP synthesis by Begley and coworkers, ^127–129 Carell and coworkers ¹³⁰ accept reported the synthesis and assay of 5R- and 5S-dinucleoside SP. Although the extent of turnover observed was extremely pocket-size, they concluded that SPL repairs merely the fiveS isomer of SP; this result was quite a surprise, as the 5South isomer would be formed in A-DNA only via interstrand cross-links and not by cross-linking adjacent thymines on the same DNA strand. A more contempo analysis of in vitro enzymatic assays on stereochemically defined SP substrates demonstrated that SPL specifically repairs but the 5 R isomer of SP. The observation that 5 R-SP, merely not v S-SP, is a substrate for SPL is consequent with the expectation that 5 R is the SP isomer produced in vivo upon UV irradiation of bacterial spore DNA. ¹³¹

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Genetic Toxicology

Joseph R. Landolph , in Encyclopedia of Toxicology (2d Edition), 2005

DNA Repair

We at present know that in bacteria, yeast, and mammalian cells, there are enzymatic systems that can repair damaged DNA. These systems are known as Deoxyribonucleic acid repair systems.

Much of the repair that takes identify in bacteria and in mammalian cells whose Dna has been damaged by chemic mutagens or by ionizing radiation, proceeds with a high degree of fidelity, and repairs the DNA damage correctly. However, a certain fraction of this repair proceeds incorrectly, and this misrepair leads to mutations. While some of this misrepair generates mutations and can be cytotoxic, a fraction of this misrepair is beneficial by generating mutations that can atomic number 82 to genetic variety in organisms and hence provides new organisms that tin can lead to development of various species.

In bacteria, nosotros at present recognize a number of DNA repair systems. The first repair system, which has been the most intensively studied and the best understood, is the organization of photoreactivation repair, or direct repair. This repair system is very efficient at repairing thymine dimers formed betwixt thymine bases in Dna by absorption of UV low-cal of 254 nm past the thymine bases. In this blazon of repair, an antenna pigment, methylene tetrahydrofolate (MTHF), absorbs most UV light of wavelength 350 nm. MTHF so transfers the free energy of this photon past Forster resonance energy transfer to reduced flavin adenine dinucleotide (FADH). FADH then transfers an electron to the thymine dimer, which decomposes information technology, returning it to its original land of two separate thymine bases in Dna. This repair takes place in the presence of the photoreactivating enzyme, which contains a pocket that binds to and holds the thymine dimer in place. Since this repair system returns the thymine dimer to its original split up thymine bases in DNA, no mutations occur during this process. Hence, this repair is said to be 'mistake-free', and information technology does not induce mutations. The photoreactivating enzyme has been cloned and sequenced, and X-ray crystallographic assay of the photoreactivating enzyme has revealed the structure of this enzyme.

A second DNA repair system in bacteria is designated excision repair. This repair system efficiently repairs DNA strands that have been irradiated with UV light or ionizing radiations, oxidatively damaged, or that have chemic-Deoxyribonucleic acid adducts in them. This system involves the steps of incision by an incision endonuclease proximate to the site of the harm, followed by excision of the damaged DNA bases by Deoxyribonucleic acid polymerase I. Next, DNA polymerase I fills in the resulting nucleotide gaps by adding nucleotides complementary to the undamaged strand, using the undamaged strand as a template. Finally, Deoxyribonucleic acid ligase seals the phosphodiester chain. This repair proceeds with a loftier caste of fidelity, and therefore only induces a very low frequency of mutations. Some authors refer to this as 'error-free' DNA repair, although a low frequency of mutations are created by this repair system.

A third blazon of DNA repair is called the SOS response. The SOS response involves the induction of two different types of DNA repair. In this situation, where there are thymine dimers in DNA due to UV irradiation of the DNA, or other Dna impairment, a normally quiescent molecule, called the rec A protease, binds to the site of this Deoxyribonucleic acid harm. The bounden of rec A to damaged Deoxyribonucleic acid causes the rec A protease to become catalytically agile. The rec A protease then binds to various molecules of the lex A repressor that are already bound to the bacterial genome. Lex A repressor molecules normally demark to the SOS boxes of genes in the genome that encode endonucleases, exonucleases, helicases, DNA polymerases, and other molecules important in Deoxyribonucleic acid repair. When the activated rec A protease binds to the Lex A repressors, this causes the Lex A repressors to autocatalytically cleave themselves. This results in the consecration of the synthesis of ∼50 protein molecules involved in Deoxyribonucleic acid repair, among them an error-prone DNA polymerase. This error-prone Deoxyribonucleic acid polymerase causes nucleotide synthesis to occur contrary the thymine dimers, with a low caste of fidelity. This leads to mutations in the DNA. In addition, during the SOS response, the rec A protease also acts as a recombinogenic enzyme. In this case, at a replication fork containing a thymine dimer, rec A-mediated recombination can occur to generate a state of affairs in which there is at to the lowest degree i skilful template for DNA synthesis on each strand of the replication fork. While allowing Deoxyribonucleic acid repair and hence DNA synthesis to proceed, rec A-mediated recombination is as well a process that proceeds with a low caste of fidelity, with fault rates of 1/1000, leading also to mutations. Similar types of Deoxyribonucleic acid be in mammalian cells.

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Genetic Toxicology

J.R. LandolphJr., in Encyclopedia of Toxicology (Third Edition), 2022

Deoxyribonucleic acid Repair in Bacteria

It was now known that in leaner, yeast, and mammalian cells, there are enzymatic systems that can repair damaged Dna. These systems are known equally Dna repair systems. Much of the Dna repair that takes place in bacteria and in mammalian cells whose DNA has been damaged by chemical mutagens or by ionizing radiations, proceeds with a loftier degree of fidelity, and repairs the DNA impairment correctly. However, a sure small fraction of this DNA repair gain incorrectly, and this misrepair leads to mutations. While some of this misrepair generates mutations and can be cytotoxic, a fraction of this misrepair is benign past generating mutations that can lead to genetic diverseness in organisms and hence provides new organisms that can lead to development of various species.

In bacteria, a number of DNA repair systems are now recognized. The first repair system, which has been the well-nigh intensively studied and the best understood, is the organisation of photoreactivation repair or directly repair. This repair system is very efficient at repairing thymine dimers containing a cyclobutane ring formed betwixt thymine bases in Deoxyribonucleic acid by absorption of UV lite of 254 nm past the thymine bases. In photoreactivation repair, an antenna paint, methylene tetrahydrofolate (MTHF), absorbs near UV calorie-free of wavelength 350 nm. MTHF so transfers the free energy of this photon past Forster resonance energy transfer to reduced flavin adenine dinucleotide (FADH). FADH then transfers an electron to the thymine dimer, which decomposes information technology, returning it to its original state of ii separate thymine bases in DNA. This repair takes identify in the presence of the photoreactivating enzyme, which contains a pocket that binds to and holds the thymine dimer in place. Since this repair system returns the thymine dimer to its original separate thymine bases in Dna, no mutations occur during this process. Hence, this repair is said to be 'error free,' and information technology does not induce mutations. The cistron that encodes the photoreactivating enzyme has been cloned and sequenced, and the photoreactivating protein has been purified and sequenced. X-ray crystallographic analysis of the photoreactivating enzyme has revealed the structure of this enzyme. It contains 2 globular domains, an NH _ii domain and a COOH domain, continued by a bridge consisting of 71 amino acids. The MTHF antenna pigment and the FADH cofactor fit in a higher place and below the amino span connecting the two globular domains. The cyclobutane pyrimidine dimer flips out from the DNA and binds to a 'pocket' in the photoreactivating enzyme. This structure easily allows the MTHF to absorb light of 340 nm, to laissez passer the free energy of this light to the FADH cofactor by Forster resonance energy transfer, and for the resultant FADH^∗ to pass an energetic electron to the cyclobutane pyrimidine dimer, causing this structure to rearrange back into two divide thymine bases, which is the original structure before thymine dimer formation.

A second DNA repair organisation in bacteria is designated excision repair. This repair system efficiently repairs Deoxyribonucleic acid strands that have been irradiated with UV light or ionizing radiations, oxidatively damaged, or that have chemical–Dna base of operations covalent adducts in them. This system involves the steps of incision past an incision endonuclease proximate to the site of the damage, followed by excision of the damaged Deoxyribonucleic acid bases by Dna polymerase I. Next, DNA polymerase I (the Arthur Kornberg enzyme) fills in the resulting nucleotide gaps by adding nucleotides complementary to the undamaged strand, using the undamaged strand as a template, and synthesizing Dna in a v′ to iii′ direction. Finally, DNA ligase seals the phosphodiester chain. This repair gain with a loftier degree of fidelity, and, therefore, only induces a very low frequency of mutations. Some authors refer to this as 'error-free' Dna repair, although a depression frequency of mutations is created by this repair organization. Every bit noted before, a low frequency of mutations is beneficial to development and is therefore acceptable.

A tertiary type of DNA repair is chosen the SOS response. The SOS response involves the induction of two different types of DNA repair. In this situation, where there are thymine dimers in DNA due to UV irradiation of the Deoxyribonucleic acid or other Dna damage, a normally quiescent molecule, called the rec A protease, binds to the site of this DNA damage. The binding of rec A to damaged Dna causes the rec A protease to become catalytically agile, and this active rec A was designated as rec A^∗. The rec A^∗ protease and so binds to Lex A repressor molecules that are already leap to diverse genes of the bacterial genome. Lex A repressor molecules normally bind to the SOS boxes of genes in the genome that encode endonucleases, exonucleases, helicases, DNA polymerases, and other molecules important in SOS repair and in postreplication recombination DNA repair. When the activated rec A protease binds to the Lex A repressors, this causes the Lex A repressors to autocatalytically cleave themselves. This results in the induction of the synthesis of ∼50 protein molecules involved in Dna repair, among them an error-decumbent Deoxyribonucleic acid polymerase. This error-prone Dna polymerase is composed of two umuD′ molecules and one umuC molecule, with the formula, umuD2′C. This mistake-prone DNA polymerase causes nucleotide synthesis to occur opposite the thymine dimers, with a low caste of allegiance. This leads to high frequencies of mutations in the Deoxyribonucleic acid, on the order of i/1000 nucleotides. This, however, is acceptable, considering otherwise, failure to repair UV-damaged DNA at the replication fork earlier the cell divides tin can impale the prison cell. In improver, during the SOS response, postreplication, recombination repair is simultaneously induced. In postreplication, recombination repair, the rec A^∗ protease also acts every bit a recombinogenic enzyme. In this case, at a replication fork containing a thymine dimer, rec A-mediated recombination can occur to generate a situation in which at that place is at least one good template for DNA synthesis on each strand of the replication fork. While assuasive Deoxyribonucleic acid repair and hence DNA synthesis to proceed, rec A-mediated recombination is besides a procedure that proceeds with a low caste of allegiance, with error rates of 1/1000, leading also to mutations.

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Introduction to Food Irradiation and Medical Sterilization

Laurence McKeen , in The Effect of Sterilization on Plastics and Elastomers (Third Edition), 2022

1.iii.5 Ionizing Radiations

Sterilization by ionizing radiation, primarily past Cobalt-60 gamma rays or electron accelerators, is a low-temperature sterilization method that has been used for a number of medical products (e.g. tissue for transplantation, pharmaceuticals, and medical devices). Gamma and electron axle (or beta) radiation is discussed in detail in the before sections on the irradiation of food. The radiation procedure is not any different for medical items, but in general only dispensable or ane-time use items are sterilized by these methods because the facilities large and expensive and are non located on hospital grounds. Major advantages of gamma and electron axle irradiation are that there are no residuals and no radioactivity remaining. Every bit soon equally the delivered dose of radiation is verified, products may be released for shipment.

In that location are no FDA-cleared ionizing radiation sterilization processes for utilize in wellness care facilities. Because of high sterilization costs, this method is an unfavorable culling to EtO and plasma sterilization in health care facilities but is suitable for large-scale sterilization.

i.3.5.1 UV Radiations

The wavelength of UV radiation ranges from 328 nm to 210 nm (3280 Å to 2100 Å). Its maximum bactericidal effect occurs at 240–280 nm. Mercury vapor lamps emit more than 90% of their radiation at 253.vii nm, which is near the maximum microbicidal activity. Inactivation of microorganisms results from destruction of nucleic acrid through induction of thymine dimers. This is discussed in Section 1.2.8. UV radiations has been employed in the disinfection of drinking h2o, air, titanium implants, and contact lenses. Bacteria and viruses are more than easily killed by UV light than are endospores.

The application of UV radiation in the health care environment (i.e. operating rooms, isolation rooms, and biologic safety cabinets) is limited to destruction of airborne organisms or inactivation of microorganisms on surfaces. There are two examples of airborne employ of UV. Figure 1.36 shows that UV lights may be mounted within air ducts to destroy airborne organisms; such an application might be constitute in operating room ventilation. Figure 1.37 shows an example of UV use in an isolation room. Table 1.ix shows the UV dose required to reduce the populations of various organisms.

Table one.9. UV Dose Required to Reduce the Population of Various Microorganisms ²³

Organisms	Energy Dosage of Ultraviolet Radiation (UV Dose) in μWs/cm² Needed for Kill Factor
Leaner	90% Reduction	99% Reduction
Bacillus anthracis – anthrax	4520	8700
Bacillus anthracis spores – anthrax spores	24,320	46,200
Bacillus megaterium sp. (spores)	2730	5200
Bacillus megaterium species (veg.)	1300	2500
Bacillus paratyphus	3200	6100
Bacillus subtilis spores	eleven,600	22,000
Bacillus subtilis	5800	11,000
Clostridium tetani	13,000	22,000
Corynebacterium diphtheriae	3370	6510
Eberthella typhosa	2,140	4100
Escherichia coli	3000	6600
Leptospira canicola – infectious jaundice	3150	6000
Micrococcus candidus	6050	12,300
Micrococcus sphaeroides	1000	fifteen,400
Mycobacterium tuberculosis	6200	x,000
Neisseria catarrhalis	4400	8500
Phytomonas tumefaciens	4400	8000
Proteus vulgaris	3000	6600
Pseudomonas aeruginosa	5500	10,500
Pseudomonas fluorescens	3500	6600
Salmonella enteritidis	4000	7600
Salmonella paratyphi – enteric fever	3200	6100
Salmonella typhosa – typhoid fever	2150	4100
Salmonella typhimurium	8000	15,200
Sarcina lutea	19,700	26,400
Serratia marcescens	2420	6160
Shigella dysenteriae – dysentery	2200	4200
Shigella flexneri – dysentery	1700	3400
Shigella paradysenteriae	1680	3400
Spirillum rubrum	4400	6160
Staphylococcus albus	1840	5720
Staphylococcus aureus	2600	6600
Staphylococcus hemolyticus	2160	5500
Staphylococcus lactis	6150	8800
Streptococcus viridans	2000	3800
Vibrio cholerae	3375	6500
Molds	90%	99%
Aspergillus flavus	60,000	99,000
Aspergillus glaucus	44,000	88,000
Aspergillus niger	132,000	330,000
Mucor racemosus A	17,000	35,200
Mucor racemosus B	17,000	35,200
Oospora lactis	5000	11,000
Penicillium expansum	thirteen,000	22,000
Penicillium roqueforti	xiii,000	26,400
Penicillium digitatum	44,000	88,000
Rhizopus nigricans	111,000	220,000
Protozoa	xc%	99%
Paramecium	11,000	20,000
Algae
Chlorella vulgaris	xiii,000	22,000
Helminthes
Nematode eggs	45,000	92,000
Virus	xc%	99%
Bacteriophage – Escherichia coli	2600	6600
Infectious hepatitis A and E	5800	8000
Flu	3400	6600
Poliovirus – Poliomyelitis	3150	6600
Tobacco mosaic	240,000	440,000
Yeast	xc%	99%
Brewers yeast	3300	6600
Mutual yeast block	6000	13,200
Saccharomyces cerevisiae	6000	thirteen,200
Saccharomyces ellipsoideus	6000	13,200
Saccharomyces spores	8000	17,600

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Photoallergens

S.C. Gad , in Encyclopedia of Toxicology (Tertiary Edition), 2022

Phototoxicity versus Photoallergenicity

From a mechanistic standpoint, calorie-free-induced dermatopathologic changes tin be divided into phototoxic and photoallergic categories. Phototoxic skin damage results from the straight interaction of irradiation with subcellular targets, whereas photoallergic reactions involve immunomodulation of cutaneous photoreactivity. Both variants require initiation by exogenous low-cal, merely subsequent cytopathologic mechanisms may be essentially dissimilar.

With phototoxicity, calorie-free may originate directly from exogenous sources, such equally the dominicus, bogus lighting, or photodynamic topical chemicals, or it may emanate from endogenous sources such as photodynamic drugs or chemicals following activation or excitation by percutaneous irradiation. Subcellular targets have not been completely characterized but may include the formation of thymine dimers, DNA–protein cantankerous-links, or photodependent oxidations. Immunologic processes are not involved in this form of photosensitivity.

With photoallergic reactions, cytopathologic events are believed to exist even more complex than with straight phototoxicity. Although many mechanistic features remain obscure, fundamental concepts include the photoactivation of endogenous or xenobiotic haptens so that they combine with cellular proteins and form a complete antigen. Subsequent immunologic reactions, especially cell-mediated hypersensitivity, complete the sensitivity process.

In dissimilarity to phototoxicity, photoallergy represents a true type IV delayed hypersensitivity reaction. Hence, although phototoxic reactions can occur with the starting time exposure to the offending chemical, photoallergy requires prior sensitization. Induction and subsequent elicitation of reactions may result from topical or systemic exposure to the amanuensis. If topical, the reactions are termed photocontact dermatitis, whereas systemic exposures are termed systemic photoallergy. In many situations, systemic photoallergy is the result of the administration of medications. Generally, the mechanisms of photocontact dermatitis and that of systemic photoallergy are the same as those for allergic contact dermatitis. In the context of photocontact dermatitis, nevertheless, UV calorie-free is necessary to convert a potential photosensitizing chemical into a hapten that elicits an allergic response.

Although precise cytopathologic mechanisms have not been established for many photosensitivity reactions, clinical and pathologic features take been extensively documented. The post-obit outline describes key diagnostic findings that serve to differentiate photosensitivity reactions from other dermatologic phenomena.

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UmuC D Lesion Bypass Deoxyribonucleic acid Polymerase 5

Fifty.A. Hawver , P.J. Beuning , in Encyclopedia of Biological Chemistry (Second Edition), 2022

Abstruse

DNA is continuously damaged, which can eventually lead to mutations and cell expiry. This damage is bypassed by Y family unit DNA polymerases which are conserved throughout evolution and have the specialized ability to re-create damaged Deoxyribonucleic acid. Escherichia coli Dna polymerase V (pol V) is a Y family polymerase composed of UmuC, which is the polymerase subunit, and the UmuD′₂ accompaniment subunit. UmuD′₂ C is known to bypass thymine–thymine dimers caused by UV radiation, besides equally abasic sites that ascend via multiple mechanisms. UmuD′ ₂C is regulated as office of the SOS response and performs potentially mutagenic translesion synthesis (TLS) to replicate damaged Deoxyribonucleic acid.

Pol V of E. coli is a member of the mistake-prone Y family unit of Dna polymerases, which accept the specialized ability to re-create damaged DNA in a process known equally 'translesion synthesis' (TLS). Politician V consists of the 48-kDa UmuC subunit, which possesses the polymerase activity, and the homodimeric accessory subunit UmuD′_two (12 kDa each), and is written as UmuD′₂C. UmuD′ results from the self-cleavage of the slightly larger protein UmuD (15 kDa). Ecology and chemical mutagens, for case, ultraviolet (UV) radiation and mitomycin C, respectively, can crusade damage to DNA and stress to cells. When this occurs, the cell initiates a response known as the 'SOS response', causing a cascade of events. The expression of the umuDC genes, encoding pol V, is induced as part of this response. This specialized polymerase bypasses common lesions from UV radiation, such as thymine–thymine (T–T) cis–syn cyclobutane pyrimidine dimers (CPD) and T–T (6–4) photoproducts, too as abasic sites. Pol V and other members of the Y family are characterized past depression processivity, low allegiance when copying undamaged Dna, and a lack of proofreading. The cofactors RecA, SSB, β processivity clamp, and γ clamp loader all facilitate TLS.

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DNA Repair Enzymes: Structure, Biophysics, and Mechanism

Kyle T. Powers , M. Todd Washington , in Methods in Enzymology, 2022

three.1 Polymerase Assays

1 of the simplest and virtually common approaches to examine the power of nonclassical polymerases to synthesize Deoxyribonucleic acid on damaged and nondamaged templates is a polymerase analysis (Fig. iiA ). In this assay, the DNA substrate is fabricated by annealing a short, ³²P-end-labeled primer strand (usually 25–thirty nucleotides in length) to a longer template strand (commonly l–75 nucleotides in length). Depending on the location of the damage in the Dna substrate, these assays may be either "standing showtime" or "running start" experiments. In standing get-go experiments, the damaged template is the first available template residue. In running start experiments, several nondamaged residues in the template are used before the enzyme encounters the harm.

In polymerase assays, the nonclassical polymerase (ordinarily 1–10 nThousand concentration) is preincubated with the DNA substrate (usually 10–50 nM concentration). The reactions are initiated with the addition of all four dNTPs (usually ten–20 μM each). Reactions are quenched afterwards various time intervals (unremarkably 2–30 min). The substrate and products of the reaction are then visualized on a denaturing polyacrylamide gel with single-nucleotide resolution—in other words, an old-fashioned sequencing gel. The pattern of gel bands contains useful information near the ability of nonclassical polymerases to bypass specific forms of DNA damage and provides potentially important clues about where kinetic barriers to nucleotide incorporation exist.

Archetype examples of the use of polymerase reactions come from studies of the ability of yeast DNA pol η to bypass two types of ultraviolet radiation-induced Deoxyribonucleic acid lesions. In the instance of cis-syn thymine–thymine dimers, politico η-catalyzed DNA synthesis was indistinguishable on the damaged and nondamaged Dna substrates ( Johnson et al., 1999). No gel bands accumulated in experiments with damaged Deoxyribonucleic acid that did not likewise accumulate in control experiments with nondamaged Deoxyribonucleic acid. This suggested that the cis-syn thymine–thymine dimer poses no additional kinetic barrier to Deoxyribonucleic acid synthesis by pol η. By contrast, in the instance of the (6-4) photoproduct, a distinct gel band accumulated at a position corresponding to incorporation opposite the 3′T of the photoproduct with no further extension products observed (Johnson, Haracska, Prakash, & Prakash, 2001). This indicated that the 6-4 photoproduct imposes a strong kinetic barrier to incorporating opposite the five′T and that Dna synthesis terminates later on incorporation opposite the 3′T.

Another apply of polymerase assays is to make up one's mind the processivity of nonclassical polymerases (Von Hippel, Fairfield, & Dolejsi, 1994; Washington, Johnson, Prakash, & Prakash, 1999). Processivity is a measure of how many nucleotides a DNA polymerase incorporates earlier dissociating from the DNA. When measuring processivity, it is necessary that the experiment be performed under "single hit" weather so that when a polymerase dissociates from the Dna template, the template volition not be engaged by another polymerase. This can be accomplished past ensuring that the DNA substrate is in large molar excess over the polymerase and that the reaction is stopped before more than twenty% of the Deoxyribonucleic acid substrates are extended. Quantification of the gel ring intensities can be used to calculate P(n), which is the probability that a polymerase that has incorporated at least northward nucleotides will incorporate additional nucleotides rather than dissociate. This is calculated using Eq. (1):

(1) $P (north) = \frac{I_{n + i} + I_{northward + 2} + \cdot \cdot \cdot}{I_{n} + I_{n + 1} + I_{due north + ii} + \cdot \cdot \cdot}$

where I _n is the intensity of the gel ring corresponding to n nucleotides incorporated, I _{due north + 1} is the intensity of the gel band corresponding to n + 1 nucleotides incorporated, and then on. Enzymes with high processivity will accept boilerplate P(northward) values very close to one, and enzymes with low processivity will take an boilerplate P(n) values less than 1. In the case of yeast DNA politico η, its processivity is low with an average P(n) value around 0.7 (Washington et al., 1999).

The major advantage of polymerase assays is their ease relative to other approaches. The major disadvantage is that they are but semiquantitative. One often sees the percentage of primer extension or the percentage of lesions bypass reported in the literature. These values are oft used to compare the abilities of an enzyme to utilize various DNA substrates. This can be problematic. For case, consider a scenario where i wants to compare the ability of a nonclassical polymerase on damaged vs nondamaged Deoxyribonucleic acid substrates. If the Thou _{one thousand} values for the two cases are significantly different, if the k _cat values for these cases are comparable, and if 1 uses dNTP concentrations that are significantly greater than the Yard _thou values (which is nearly ever the case in such assays), then the percentage of lesion bypass will be similar in these two cases despite significant differences in the efficiency of damage bypass between the two reactions. Thus, extracting meaningful information near the efficiency of harm bypass from these semiquantitative assays is highly problematic. The easiest style to compare the efficiencies of two reactions is to use steady-state kinetics.

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Carcinogenesis

F.R. de Gruijl , H.N. Ananthaswamy , in Comprehensive Toxicology, 2010

14.09.ii.4 UV-Induced Mutation

UV radiation induces unique dimeric lesions in the DNA, and these give rise to rather unique mutations in genes. Both pyrimidine dimers and (6–4) photoproducts take been shown to be mutagenic in Escherichia coli and mammalian cells (Brash 1988; Mitchell and Nairn 1989; Sage 1993). These two lesions disrupt cellular processes past obstructing the DNA and RNA synthesizing machineries and lead to the incorporation of wrong bases into the genetic fabric. These types of mistakes often event in mutation leading to loss or inappropriate expression of the affected genes. UV radiation induces predominantly C to T and CC to TT transitions at dipyrimidine sequences, which have become known as the 'UV signature mutations' (Brash 1988). These mutations are hypothesized to arise during semiconservative replication of the Deoxyribonucleic acid due to default incorporation of A residues at noninstructional sites, the 'A dominion' (Advised et al. 1991). When the DNA polymerase comes across lesions in the Deoxyribonucleic acid template that information technology cannot interpret, the replication complex tries to execute 'translesional synthesis' by switching between unlike types of polymerases, where the polymerase η (defective in XP variant) inserts A residues opposite dimerized pyrimidines (Matsuda et al. 2000 ). Thus, thymine–thymine dimers, the virtually frequent UV-induced lesions in the DNA, do non give ascension to any mutation considering the normal complementary base to T is A. The occurrence of C toT and CC to TT mutations at cytosine–cytosine sites, every bit predicted by the A dominion, has been really demonstrated in homo cells ( Bredberg et al. 1986). In addition, the presence of UV signature mutations in the p53 tumor suppressor gene in human being peel cancers and in mouse peel cancers induced by UV radiation has been well documented (Brash et al. 1991; Dumaz et al. 1993; Dumaz et al. 1997; Greenblatt et al. 1994; Kanjilal et al. 1993; Kress et al. 1992; Moles et al. 1993; Nakazawa et al. 1994; Nelson et al. 1994; Pierceall et al. 1991b; Rady et al. 1992; Sato et al. 1993; van der Riet et al. 1994; Ziegler et al. 1993, 1994).

Every bit mentioned earlier, UV-A radiation and visible lite induce the formation of reactive oxygen species in cells leading to the production of singlet oxygen, hydrogen peroxide, and other radicals. One of the base of operations alterations produced by singlet oxygen is the oxidation of guanine residues such as 8-hydroxyguanine. DNA damage of this kind in the genome induces G to T transversions by mispairing with A (Cheng et al. 1992; Moriya et al. 1991; Wood et al. 1990). In Chinese hamster ovary (CHO) cells UV-A radiation was establish to induce many T to G transversions (Drobetsky et al. 1995), which could exist due to formation of 8-oxo-dGTP (viii-oxo-seven, 8-dihydro-two′-deoxyguanosine v′-triphosphate) in the nucleoside pool, which is then erroneously incorporated in a newly synthesized DNA strand opposite an A in the template strand (Kobayashi et al. 1998). Singlet oxygen also produces G to C and Grand to A substitutions (Piette 1991). Hydrogen peroxide, produced intracellularly past solar UV radiation, as well induces a variety of substitutions at G:C base pairs (Moraes et al. 1989). Reid and Loeb (1993) demonstrated that reactive oxygen species such as hydroxyl radicals could induce CC to TT transitions in Escherichia coli, although the frequency of these mutations was far less than that of unmarried-base substitutions.

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