Which Repair Process In E. Coli Uses Visible Light To Repair Thymine Dimers

Enzymatic recognition of radiation-produced oxidative DNA lesion. Molecular dynamics approach

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

10.5.2 Thymine dimer (5,6 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 skin cancer. It is formed as a covalently bonded complex of two adjacent thymines on a single strand of DNA. This damage is very frequent but almost 90 percent of TDs are repaired within a short time of the order of minutes, and only a few are experimentally observable and originate future changes at the 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 MD simulation indicate that this lesion does not disrupt the double helical structure 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 bending at the dimer site which is originated by two covalent bonds C(5)-C(5) and C(6)-C(6) between adjacent thymines forming thymine dimer (Fig. 10.2). This bending is supposed to discriminate lesion from undamaged DNA segment and to originate conformation that facilitates the formation of a DNA–enzyme complex by generating complementary structural shapes of repair enzymes and bent DNA.

Thymine dimer excision repair is initiated by E. coli endonuclease V of bacteriophage T4 that slides on non-target sequences and progressively incises at all dimers within the DNA molecule. This enzyme binds to the DNA double strand in a two-step process: at first it scans non-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 V to thymine dimer lesioned DNA was simulated using the MD method. Considering the limitations arising from the simulations of large systems and requirements for CPU time, instead of the whole enzyme a small isolated part that included a catalytic center was subjected to the simulations. The part 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 role in the cleavage of the N-glycosyl bond in DNA during enzymatic repair process [27, 28], is surrounded by amino acids Arg-3, 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, eight 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 NH₂ terminus, Thr-2 and Arg-3, were selected to form the simulated part of the enzyme. Amino acids Thr-2, Arg-22, Glu-23 and Arg-26 form the catalytic center that is active in the incision of the thymine dimmer during the repair process and together with other six selected amino acids: Arg-3, Glu-20, Tyr-21, Leu-24, Pro-25 and Val-27, are located at the central part of a concave site of the enzyme and may be easily exposed to the DNA surface.

MD simulation showed that after nearly 100 ps of the MD simulation, the catalytic part 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 area between DNA and the enzyme it can be observed that there is an intensive interaction arising from the close 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 can also be noted that Arg-22 and Arg-26 contribute to the recognition and stability of the complex structure. Glu-23, cleaving the N-glycosyl bond in the repair process, is located close to the C5′ atom. These observations are in good agreement with crystal structure, where side chains of Arg-22 and Arg-26 form stacking contact with the DNA [29].

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

In this simulation only a relatively small 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 energy in the recognition process, as will be discussed later 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 calculation on the thymine dimer in the previous section showed that the ring fusion at the C5 and C6 atoms of two thymine bases created the four-member cyclobutane puckered ring. The same feature 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 two 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 ring and axial 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.e., hyperconjugation). The highest occupied molecular orbital (HOMO) of the neutral T<>T is localized on the C6A-C6B bond.

After ionization, the SOMO orbital of TTp-1 is localized on the C6A-C6B bond (see Figure 8(a)), and the bond is lengthened (see Table 8) compared to the neutral species, as the bond is now weaker due to the single electron occupancy. The four-member ring in TTp-1 is puckered (see 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 atom than that on the C6B atom (see Table 7) is probably caused by this effect. The puckering of the cyclobutane ring is responsible for the difference in the electronic structure in the two thymine rings and plays an important role in determining the dissociation path as will be described later.

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Enzymes and Enzyme Mechanisms

Eric M. Shepard , Joan B. Broderick , in Comprehensive Natural Products II, 2010

8.17.3.4 DNA Repair: SPL

The major photoproduct in UV-irradiated Bacillus spore DNA is a unique thymine dimer called spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine). ^108–110 In contrast, UV irradiation of DNA in most growing cells produces primarily cyclobutane pyrimidine dimers as well as the 6,4-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 as small, acid-soluble proteins (SASPs). ^112–115 It has been proposed that binding of SASPs to DNA promotes a structural change and a change 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 can block replication and transcription or can result in mutations if transcription proceeds past the region of the dimer. Repair of these dimers, therefore, is critical in order to avoid mutations, and thus is the key 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 DNA photolyase. ^111,119 However, photoreactivation has been shown to be absent in many species, including Bacillus, suggesting that alternate means of pyrimidine dimer repair might be found. ^109,120

The enzyme SPL is the first identified nonphotoactivatable pyrimidine dimer lyase and it specifically targets SP and cleaves it into two 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 Fe–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 by direct H-atom abstraction 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 as the radical SAM enzyme that utilizes SAM as a cofactor. Following on the initial reports of SP synthesis by Begley and coworkers, ^127–129 Carell and coworkers ¹³⁰ have reported the synthesis and assay of 5R- and 5S-dinucleoside SP. Although the extent of turnover observed was extremely small, they concluded that SPL repairs only the 5S isomer of SP; this result was quite a surprise, as the 5S 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 recent analysis of in vitro enzymatic assays on stereochemically defined SP substrates demonstrated that SPL specifically repairs only the 5 R isomer of SP. The observation that 5 R-SP, but not 5 S-SP, is a substrate for SPL is consistent 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 (Second Edition), 2005

DNA Repair

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

Much of the 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 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 lead to genetic diversity in organisms and hence provides new organisms that can lead to evolution of various species.

In bacteria, we now recognize a number of DNA repair systems. The first repair system, which has been the most intensively studied and the best understood, is the system of photoreactivation repair, or direct repair. This repair system is very efficient at repairing thymine dimers formed between thymine bases in DNA by absorption of UV light of 254 nm by the thymine bases. In this type of repair, an antenna pigment, methylene tetrahydrofolate (MTHF), absorbs near UV light of wavelength 350 nm. MTHF then transfers the energy of this photon by Forster resonance energy transfer to reduced flavin adenine dinucleotide (FADH). FADH then transfers an electron to the thymine dimer, which decomposes it, returning it to its original state 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 separate thymine bases in DNA, no mutations occur during this process. Hence, this repair is said to be 'error-free', and it does not induce mutations. The photoreactivating enzyme has been cloned and sequenced, and X-ray crystallographic analysis 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 chemical-DNA adducts in them. This system involves the steps of incision by an incision endonuclease proximate to the site of the damage, followed by excision of the damaged DNA bases by DNA 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, DNA ligase seals the phosphodiester chain. This repair proceeds with a high 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 low frequency of mutations are created by this repair system.

A third type 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 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 active. 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 bind to the SOS boxes of genes in the genome that encode endonucleases, exonucleases, helicases, DNA polymerases, and other molecules important in 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-prone DNA polymerase. This error-prone DNA polymerase causes nucleotide synthesis to occur opposite the thymine dimers, with a low degree 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 situation in which there is at least one good template for DNA synthesis on each strand of the replication fork. While allowing DNA repair and hence DNA synthesis to proceed, rec A-mediated recombination is also a process that proceeds with a low degree of fidelity, with error rates of 1/1000, leading also to mutations. Similar types of DNA exist in mammalian cells.

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Genetic Toxicology

J.R. LandolphJr., in Encyclopedia of Toxicology (Third Edition), 2014

DNA Repair in Bacteria

It was now known that in bacteria, yeast, and mammalian cells, there are enzymatic systems that can repair damaged DNA. These systems are known as 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 high degree of fidelity, and repairs the DNA damage correctly. However, a certain small fraction of this DNA 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 lead to genetic diversity in organisms and hence provides new organisms that can lead to evolution of various species.

In bacteria, a number of DNA repair systems are now recognized. The first repair system, which has been the most intensively studied and the best understood, is the system of photoreactivation repair or direct repair. This repair system is very efficient at repairing thymine dimers containing a cyclobutane ring formed between thymine bases in DNA by absorption of UV light of 254 nm by the thymine bases. In photoreactivation repair, an antenna pigment, methylene tetrahydrofolate (MTHF), absorbs near UV light of wavelength 350 nm. MTHF then transfers the energy of this photon by Forster resonance energy transfer to reduced flavin adenine dinucleotide (FADH). FADH then transfers an electron to the thymine dimer, which decomposes it, returning it to its original state 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 separate thymine bases in DNA, no mutations occur during this process. Hence, this repair is said to be 'error free,' and it does not induce mutations. The gene 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 two globular domains, an NH ₂ domain and a COOH domain, connected by a bridge consisting of 71 amino acids. The MTHF antenna pigment and the FADH cofactor fit above and below the amino bridge 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 pass the 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 separate thymine bases, which is the original structure before thymine dimer formation.

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 chemical–DNA base covalent adducts in them. This system involves the steps of incision by an incision endonuclease proximate to the site of the damage, followed by excision of the damaged DNA 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 5′ to 3′ direction. Finally, DNA ligase seals the phosphodiester chain. This repair proceeds with a high 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 low frequency of mutations is created by this repair system. As noted before, a low frequency of mutations is beneficial to evolution and is therefore acceptable.

A third type 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 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 active, and this active rec A was designated as rec A^∗. The rec A^∗ protease then binds to Lex A repressor molecules that are already bound to various 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-prone DNA polymerase. This error-prone DNA polymerase is composed of two umuD′ molecules and one umuC molecule, with the formula, umuD2′C. This error-prone DNA polymerase causes nucleotide synthesis to occur opposite the thymine dimers, with a low degree of fidelity. This leads to high frequencies of mutations in the DNA, on the order of 1/1000 nucleotides. This, however, is acceptable, because otherwise, failure to repair UV-damaged DNA at the replication fork before the cell divides can kill the cell. In addition, during the SOS response, postreplication, recombination repair is simultaneously induced. In postreplication, recombination repair, 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 situation in which there is at least one good template for DNA synthesis on each strand of the replication fork. While allowing DNA repair and hence DNA synthesis to proceed, rec A-mediated recombination is also a process that proceeds with a low degree of fidelity, 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), 2012

1.3.5.1 UV Radiation

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 253.7 nm, which is near the maximum microbicidal activity. Inactivation of microorganisms results from destruction of nucleic acid through induction of thymine dimers. This is discussed in Section 1.2.8. UV radiation has been employed in the disinfection of drinking water, air, titanium implants, and contact lenses. Bacteria and viruses are more 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 use of UV. Figure 1.36 shows that UV lights may be mounted within air ducts to destroy airborne organisms; such an application might be found in operating room ventilation. Figure 1.37 shows an example of UV use in an isolation room. Table 1.9 shows the UV dose required to reduce the populations of various organisms.

Table 1.9. 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
Bacteria	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	11,600	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	15,400
Mycobacterium tuberculosis	6200	10,000
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	13,000	22,000
Penicillium roqueforti	13,000	26,400
Penicillium digitatum	44,000	88,000
Rhizopus nigricans	111,000	220,000
Protozoa	90%	99%
Paramecium	11,000	20,000
Algae
Chlorella vulgaris	13,000	22,000
Helminthes
Nematode eggs	45,000	92,000
Virus	90%	99%
Bacteriophage – Escherichia coli	2600	6600
Infectious hepatitis A and E	5800	8000
Influenza	3400	6600
Poliovirus – Poliomyelitis	3150	6600
Tobacco mosaic	240,000	440,000
Yeast	90%	99%
Brewers yeast	3300	6600
Common yeast cake	6000	13,200
Saccharomyces cerevisiae	6000	13,200
Saccharomyces ellipsoideus	6000	13,200
Saccharomyces spores	8000	17,600

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Photoallergens

S.C. Gad , in Encyclopedia of Toxicology (Third Edition), 2014

Phototoxicity versus Photoallergenicity

From a mechanistic standpoint, light-induced dermatopathologic changes can be divided into phototoxic and photoallergic categories. Phototoxic skin damage results from the direct interaction of irradiation with subcellular targets, whereas photoallergic reactions involve immunomodulation of cutaneous photoreactivity. Both variants require initiation by exogenous light, but subsequent cytopathologic mechanisms may be substantially different.

With phototoxicity, light may originate directly from exogenous sources, such as the sun, artificial 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 cross-links, or photodependent oxidations. Immunologic processes are not involved in this form of photosensitivity.

With photoallergic reactions, cytopathologic events are believed to be even more complex than with direct 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 contrast to phototoxicity, photoallergy represents a true type IV delayed hypersensitivity reaction. Hence, although phototoxic reactions can occur with the first exposure to the offending chemical, photoallergy requires prior sensitization. Induction and subsequent elicitation of reactions may result from topical or systemic exposure to the agent. 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, however, UV light 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 have been extensively documented. The following outline describes key diagnostic findings that serve to differentiate photosensitivity reactions from other dermatologic phenomena.

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UmuC D Lesion Bypass DNA Polymerase V

L.A. Hawver , P.J. Beuning , in Encyclopedia of Biological Chemistry (Second Edition), 2013

Abstract

DNA is continuously damaged, which can eventually lead to mutations and cell death. This damage is bypassed by Y family DNA polymerases which are conserved throughout evolution and have the specialized ability to copy damaged DNA. Escherichia coli DNA polymerase V (pol V) is a Y family polymerase composed of UmuC, which is the polymerase subunit, and the UmuD′₂ accessory subunit. UmuD′₂ C is known to bypass thymine–thymine dimers caused by UV radiation, as well as abasic sites that arise via multiple mechanisms. UmuD′ ₂C is regulated as part of the SOS response and performs potentially mutagenic translesion synthesis (TLS) to replicate damaged DNA.

Pol V of E. coli is a member of the error-prone Y family of DNA polymerases, which have the specialized ability to copy damaged DNA in a process known as 'translesion synthesis' (TLS). Pol V consists of the 48-kDa UmuC subunit, which possesses the polymerase activity, and the homodimeric accessory subunit UmuD′₂ (12 kDa each), and is written as UmuD′₂C. UmuD′ results from the self-cleavage of the slightly larger protein UmuD (15 kDa). Environmental and chemical mutagens, for example, ultraviolet (UV) radiation and mitomycin C, respectively, can cause 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, as well as abasic sites. Pol V and other members of the Y family are characterized by low processivity, low fidelity 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, 2017

3.1 Polymerase Assays

One of the simplest and most common approaches to examine the ability of nonclassical polymerases to synthesize DNA on damaged and nondamaged templates is a polymerase assay (Fig. 2A ). In this assay, the DNA substrate is made by annealing a short, ³²P-end-labeled primer strand (usually 25–30 nucleotides in length) to a longer template strand (usually 50–75 nucleotides in length). Depending on the location of the damage in the DNA substrate, these assays may be either "standing start" or "running start" experiments. In standing start 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 damage.

In polymerase assays, the nonclassical polymerase (usually 1–10 nM concentration) is preincubated with the DNA substrate (usually 10–50 nM concentration). The reactions are initiated with the addition of all four dNTPs (usually 10–20 μM each). Reactions are quenched after various time intervals (usually 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 about 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.

Classic 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 DNA lesions. In the case of cis-syn thymine–thymine dimers, pol 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 DNA that did not also accumulate in control experiments with nondamaged DNA. This suggested that the cis-syn thymine–thymine dimer poses no additional kinetic barrier to DNA synthesis by pol η. By contrast, in the case 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 5′T and that DNA synthesis terminates after incorporation opposite the 3′T.

Another use of polymerase assays is to determine 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 before dissociating from the DNA. When measuring processivity, it is necessary that the experiment be performed under "single hit" conditions so that when a polymerase dissociates from the DNA template, the template will not be engaged by another polymerase. This can be accomplished by ensuring that the DNA substrate is in large molar excess over the polymerase and that the reaction is stopped before more than 20% of the DNA substrates are extended. Quantification of the gel band intensities can be used to calculate P(n), which is the probability that a polymerase that has incorporated at least n nucleotides will incorporate additional nucleotides rather than dissociate. This is calculated using Eq. (1):

(1) $P (n) = \frac{I_{n + 1} + I_{n + 2} + \cdot \cdot \cdot}{I_{n} + I_{n + 1} + I_{n + 2} + \cdot \cdot \cdot}$

where I _n is the intensity of the gel band corresponding to n nucleotides incorporated, I _{n + 1} is the intensity of the gel band corresponding to n + 1 nucleotides incorporated, and so on. Enzymes with high processivity will have average P(n) values very close to 1, and enzymes with low processivity will have an average P(n) values less than 1. In the case of yeast DNA pol 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 only semiquantitative. One often sees the percentage of primer extension or the percentage of lesions bypass reported in the literature. These values are frequently used to compare the abilities of an enzyme to utilize various DNA substrates. This can be problematic. For example, consider a scenario where one wants to compare the ability of a nonclassical polymerase on damaged vs nondamaged DNA substrates. If the K _m values for the two cases are significantly different, if the k _cat values for these cases are comparable, and if one uses dNTP concentrations that are significantly greater than the K _m values (which is nearly always 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 about the efficiency of damage bypass from these semiquantitative assays is highly problematic. The easiest way 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

14.09.2.4 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 have 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 by obstructing the DNA and RNA synthesizing machineries and lead to the incorporation of wrong bases into the genetic material. These types of mistakes often result 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 DNA due to default incorporation of A residues at noninstructional sites, the 'A rule' (Brash et al. 1991). When the DNA polymerase comes across lesions in the DNA template that it cannot interpret, the replication complex tries to execute 'translesional synthesis' by switching between different 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 most frequent UV-induced lesions in the DNA, do not give rise to any mutation because the normal complementary base to T is A. The occurrence of C toT and CC to TT mutations at cytosine–cytosine sites, as predicted by the A rule, has been actually demonstrated in human cells ( Bredberg et al. 1986). In addition, the presence of UV signature mutations in the p53 tumor suppressor gene in human skin cancers and in mouse skin 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).

As mentioned before, UV-A radiation and visible light 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 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 found to induce many T to G transversions (Drobetsky et al. 1995), which could be due to formation of 8-oxo-dGTP (8-oxo-7, 8-dihydro-2′-deoxyguanosine 5′-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 G to A substitutions (Piette 1991). Hydrogen peroxide, produced intracellularly by solar UV radiation, also 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 single-base substitutions.

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