UV Photoproducts

In contrast to TT dimers, TT (6-4) photoproducts cannot be bypassed by Polq alone in vitro. Instead, Polq is able to insert a G opposite the 3 T of the TT (6-4) photoproduct before aborting DNA synthesis. The resulting intermediate of translesion synthesis is a substrate for extension synthesis by Pol . Coordination between these two polymerases could therefore achieve bypass of TT (6-4) photoproducts by the two-polymerase two-step mechanism of translesion synthesis. This indeed occurs in yeast cells and is the major mechanism of G mis-insertion opposite the 3  

Polr performs error-prone translesion synthesis opposite (+)- and (-)-trans-anti-BPDE-A -dG DNA adducts by predominantly inserting A opposite the lesion in vitro. This polymerase is more active in response to the former isomeric lesion. In yeast cells, Polr, Pol , and Revl are all required for G - T transvertion mutations. The likely mechanism is A insertion opposite the lesion by Polr followed by extension synthesis by Pol . Revl probably plays a noncatalytic role in such a mutagenic bypass of the BPDE lesions. 

10d AAF-dG Adducts. Based on forward mutation assays, the major mutations induced by AAF DNA adducts are frameshift mutations in yeast and human cells. In yeast, the Pol mutagenesis pathway plays a major role in error-prone translesion synthesis of AAF-dG adducts. Consistent with these in vivo results, yeast Pol is able to perform limited translesion synthesis across from an AAF-dG adduct in vitro, mis-inserting a G opposite the lesion. Furthermore, Pol is also capable of extension synthesis from opposite the AAF-dG adduct. The REV1 dCMP transferase, on the other hand, is essentially inactive in response to a template AAF-dG adduct. Fluman PoIk is capable of error-prone translesion synthesis in vitro, inserting T or C at similar frequencies and A at a lower frequency opposite the AAF-dG adduct. 

Efficient error-free nucleotide insertion opposite an AAF-dG adduct can be catalyzed by Polr in vitro. The human Polr is more efficient in subsequent extension synthesis as compared to the yeast Polq. If the error-free translesion synthesis activity of Polr is utilized in cells in response to AAF-dG adducts, this polymerase would function to suppress AAF-induced mutagenesis. In one study with yeast cells, both an error-free bypass role and a frameshift mutagenesis role of Polq were reported. Hence, it is still unknown about the contribution of Polq to AAF-induced mutagenesis. Opposite a template AAF-dG adduct, human Poll is able to insert predominantly a C in vitro. Subsequent extension synthesis, however, was not observed. 

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Cells have substantial chemical defenses against the UV photoproducts produced in seawater and intracellular fluids. Many organisms have antioxidants (e.g., carotenoids, ascorbate, tocopherols, anthocyanins, and tridentatols) that quench photo-oxidative reactions.64-67 Cells also have enzymes (e.g., catalase and superoxide dismutase) that can counteract the oxidative nature of peroxides and other radicals.26 Some compounds, such as the UV-absorbing pigment melanin, can act as both optical filter and antioxidant.68 The MAA mycosporine-glycine (Figure 15.3) functions in a similar dual capacity.69 The role of UV-mediated reactions in seawater relative to biological effects is an important current area of study. 

Figure 9-3. Structures of the UV photoproducts of the pyrimidine nucleobases. See text for details...

G. R Pfeifer, Formation and processing of UV photoproducts effects of DNA sequence and chromatin environment. Photochemistry Photobiology 65(2), 270-283 (1997). 

Howard-Flanders P, Boyce RP.Simson E, Theriot L (1962) A genetic locus in E.coli K12 that controls the reactivation of UV-photoproducts associated with thymine in DNA. Proc Natl Acad Sci USA 48 2109-15... 

Although Pol V replicates undamaged templates with relatively low fidelity (10 3 to 10-4) [76], one striking quality is Pol V s ability to accurately bypass UV photoproducts (e.g., inserting dATP opposite thymine-thymine (TT) cyclobutane pyrimidine dimers (CPDs) [76]). Analysis of insertion tendencies opposite a variety of adducts/lesions led to the observation that Pol V seems to have two insertion modes (i) correct dNTP insertion and (ii) default dATP insertion [37]. UV light is a frequently encountered form of DNA damage for which a translesion synthesis DNA polymerase might be important and since TT CPDs are the major UV lesion... 

Iwai, S and Sale, J.E. (2008) REV1 restrains DNA polymerase C, to ensure frame fidelity during translesion synthesis of UV photoproducts in vivo. Nucleic Acids Res., 36, 6767-6780. 

The (6-4) photoproduct is the second most abundant lesion induced in DNA by UV light, constituting 10%-20% of total UV photoproducts (Taylor, 1994). In contrast to cyclobutane pyrimidine dimers that are formed from the excited triplet state of pyrimidines, the (6-4) photoproducts are formed from the pyrimidine excited singlet state. In the (6-4) photoproduct, the C6 of the 5 pyrimidine makes a sigma bond with the C4 of the 3 pyrimidine, and the —OH (or —NH2) group at the C4... 

In addition to UV photoproducts, a variety of DNA lesions that result from treatment with chemical mutagens has also been examined as being potential substrates for Pol/. AAF-guanine was found by Guo et al (2001)... 

Sakofsky, C.J., Runck, L.A., and Grogan, D.W. (2011) Sulfolobus mutants, generated via PCR products, which lack putative enzymes of UV photoproduct repair. Archaea, 2011, 864015. 

The study of photo lyase began with the hrst description of photoreactivation in S. griseus in 1949. Photoreactivation is the reversal of the harmful and mutagenic effects of ultraviolet irradiation by blue-light, and in 1958, the enzyme responsible for this phenomenon, photolyase, was discovered. Photolyase uses blue-light energy to repair the two major UV photoproducts in DNA, cyclobutane pyrimidine dimers... 

The described UV photolesions are the best-studied photoproducts so far. We can, however, be sure that many more UV-induced lesions exist, which await structural characterization. Recently, CHvio et al. isolated a new photoproduct after UV irradiation of an amide-linked thymidyl-thymidine. The UV photoproduct is an interesting 2-imidazolone-(5—4)-pyrimidone adduct, shown in Scheme 7. It is yet unclear if similar photoproducts are also formed in DNA. 

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