Identification of the different intermediates in the interaction of (A)BC excinuclease with its substrates by DNase I footprinting on two uniquely modified oligonucleotides

(A)BC excinuclease is the enzymatic activity resulting from the joint actions of UvrA, UvrB and UvrC proteins of Escherichia coli. The enzyme removes from DNA many types of adducts of dissimilar structures with different efficiencies. To understand the mechanism of substrate recognition and the basis of enzyme specificity, we investigated the interactions of the three subunits with two synthetic substrates, one containing a psoralen-thymine monoadduct and the other a thymine dimer. Using DNase I as a probe, we found that UvrA makes a 33 base-pair footprint around the psoralen-thymine adduct and that UvrA-UvrB make a 45 base-pair asymmetric footprint characterized by a hypersensitive site 11 nucleotides 5' to the adduct and protection mostly on the 3' side of the damage. Conditions that favor dissociation of UvrA from the UvrA-UvrB-DNA complex, such as addition of excess undamaged DNA to the reaction mixture, resulted in the formation of a 19 base-pair UvrB footprint. In contrast, a thymine dimer in a similar sequence context failed to elicit a UvrA, a UvrA-UvrB or UvrB footprint and gave rise to a relatively weak DNase I hypersensitive site typical of a UvrA-UvrB complex. Dissociation of UvrA from the UvrA-UvrB-DNA complex stimulated the rate of incision of both substrates upon addition of UvrC, leading us to conclude that UvrA is not a part of the incision complex and that it actually interferes with incision. The extent of incision of the two substrates upon addition of UvrC (70% for the psoralen adduct and 20% for the thymine dimer) was proportional to the extent of formation of the UvrA-UvrB-DNA (i.e. UvrB-DNA) complex, indicating that substrate discrimination occurs at the preincision step.     

Cloning, sequencing, expression and characterization of DNA photolyase from Salmonella typhimurium.

We have cloned the phr gene that encodes DNA photolyase from Salmonella typhimurium by in vivo complementation of Escherichia coli phr gene defect. The S.typhimurium phr gene is 1419 base pairs long and the deduced amino acid sequence has 80% identity with that of E. coli photolyase. We expressed the S.typhimurium phr gene in E.coli by ligating the E.coli trc promoter 5' to the gene, and purified the enzyme to near homogeneity. The apparent molecular weight of S.typhimurium photolyase is 54,000 dalton as determined by SDS-polyacrylamide gel electrophoresis, which is consistent with the calculated molecular weight of 53,932 dalton from the deduced phr gene product. S.typhimurium photolyase is purple-blue in color with near UV-visible absorption peaks at 384, 480, 580, and 625 nm and a fluorescence peak at 470 nm. From the characteristic absorption and fluorescence spectra and reconstitution experiments, S.typhimurium photolyase appears to contain flavin and methenyltetrahydrofolate as chromophore-cofactors as do the E.coli and yeast photolyases. Thus, S.typhimurium protein is the third folate class photolyase to be cloned and characterized to date. The binding constant of S.typhimurium photolyase to thymine dimer in DNA is kD = 1.6 x 10(-9) M, and the quantum yield of photorepair at 384 nm is 0.5.     

Utilization of DNA photolyase, pyrimidine dimer endonucleases, and alkali hydrolysis in the analysis of aberrant ABC excinuclease incisions adjacent to UV-induced DNA photoproducts.

ABC excinuclease of Escherichia coli removes 6-4 photoproducts and pyrimidine dimers from DNA by making two single strand incisions, one 8 phosphodiester bonds 5' and another 4 or 5 phosphodiester bonds 3' to the lesion. We describe in this communication a method, which utilizes DNA photolyase from E. coli, pyrimidine dimer endonucleases from M. luteus and bacteriophage T4, and alkali hydrolysis, for analyzing the ABC excinuclease incision pattern corresponding to each of these photoproducts in a DNA fragment. On occasion, ABC excinuclease does not incise DNA exclusively 8 phosphodiester bonds 5' or 4 or 5 phosphodiester bonds 3' to the photoproduct. Both the nature of the adduct (6-4 photoproduct or pyrimidine dimer) and the sequence of neighboring nucleotides influence the incision pattern of ABC excinuclease. We show directly that photolyase stimulates the removal of pyrimidine dimers (but not 6-4 photoproducts) by the excinuclease. Also, photolyase does not repair CC pyrimidine dimers efficiently while it does repair TT or TC pyrimidine dimers.     

Role of enzyme-bound 5,10-methenyltetrahydropteroylpolyglutamate in catalysis by Escherichia coli DNA photolyase

DNA photolyase catalyzes the photoreversal of pyrimidine dimers. The enzymes from Escherichia coli and yeast contain a flavin chromophore and a folate cofactor, 5,10-methenyltetrahydropteroylpolyglutamate. E. coli DNA photolyase contains about 0.3 mol of folate/mol flavin, whereas the yeast photolyase contains the full complement of folate. E. coli DNA photolyase is reconstituted to a full complement of the folate by addition of 5,10-methenyltetrahydrofolate to cell lysates or purified enzyme samples. The reconstituted enzyme displays a higher photolytic cross section under limiting light. Treatment of photolyase with sodium borohydride or repeated camera flashing results in the disappearance of the absorption band at 384 nm and is correlated with the formation of modified products from the enzyme-bound 5,10-methenyltetrahydrofolate. Photolyase modified in this manner has a decreased photolytic cross section under limiting light. Borohydride reduction results in the formation of 5,10-methylenetetrahydrofolate and 5-methyltetrahydrofolate, both of which are released from the enzyme. Repeated camera flashing results in photodecomposition of the enzyme-bound 5,10-methenyltetrahydrofolate and release of the decomposition products. Finally, it is observed that photolyase binds 10-formyltetrahydrofolate and appears to cyclize it to form the 5,10-methenyltetrahydrofolate chromophore.     

Sequences of the E. coli uvrC gene and protein

We have determined the sequence of a 2400 bp region of E. coli chromosomal DNA containing the uvrC gene. The coding region of uvrc is 2267 bp in length, encodes a polypeptide with a calculated molecular weight of 66,038 daltons, and is preceded by a typical E. coli ribosome binding site. By constructing deletion derivatives we have established that a uvrC promoter lies within the 113 bp region preceding the translational start of uvrC. The codon usage in uvrC is strongly biased in favor of codons used infrequently in E. coli, which may contribute to the relatively low intracellular concentration of uvrC protein.     

Roles of FAD and 8-hydroxy-5-deazaflavin chromophores in photoreactivation by Anacystis nidulans DNA photolyase.

DNA photolyase from the cyanobacterium Anacystis nidulans contains two chromophores, flavin adenine dinucleotide (FADH2) and 8-hydroxy-5-deazaflavin (8-HDF) (Eker, A. P. M., Kooiman, P., Hessels, J. K. C., and Yasui, A. (1990) J. Biol. Chem. 265, 8009-8015). While evidence exists that the flavin chromophore (in FADH2 form) can catalyze photorepair directly and that the 8-HDF chromophore is the major photosensitizer in photoreactivation it was not known whether 8-HDF splits pyrimidine dimer directly or indirectly through energy transfer to FADH2 at the catalytic center. We constructed a plasmid which over-produces the A. nidulans photolyase in Escherichia coli and purified the enzyme from this organism. Apoenzyme was prepared and enzyme containing stoichiometric amounts of either or both chromophores was reconstituted. The substrate binding and catalytic activities of the apoenzyme (apoE), E-FADH2, E-8-HDF, E-FAD(ox)-8-HDF, and E-FADH2-8-HDF were investigated. We found that FAD is required for substrate binding and catalysis and that 8-HDF is not essential for binding DNA, and participates in catalysis only through energy transfer to FADH2. The quantum yields of energy transfer from 8-HDF to FADH2 and of electron transfer from FADH2 to thymine dimer are near unity.     

Determination of rates and yields of interchromophore (folate----flavin) energy transfer and intermolecular (flavin----DNA) electron transfer in Escherichia coli photolyase by time-resolved fluorescence and absorption spectroscopy

Escherichia coli DNA photolyase, which photorepairs cyclobutane pyrimidine dimers, contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and 5,10-methenyltetrahydrofolate (MTHF). Previous work has shown that MTHF is the primary photoreceptor which transfers energy to the FADH2 cofactor; the FADH2 singlet excited state then repairs the photodimer by electron transfer. In this study, we have determined the rate constants for these photophysical processes by time-resolved fluorescence and absorption spectroscopy. From time-resolved fluorescence, we find that energy transfer from MTHF to FADH2 and FADH degrees occurs at rates of 4.6 x 10(9) and 3.0 x 10(10) s-1, respectively, and electron transfer from FADH2 to a pyrimidine dimer occurs at a rate of 5.5 x 10(9) s-1. Using F?rster theory for long-range energy transfer and assuming K2 = 2/3, the interchromophore distances were estimated to be 22 A in the case of the MTHF-FADH2 pair and 21 A for the MTHF-FADH degrees pair. Picosecond absorption spectroscopy identified an MTHF single state which decays to yield the first excited singlet state of FADH2. The lifetimes of MTHF and FADH2 singlets and the rates of interchromophore energy transfer, as well as the rate of electron transfer from FADH2 to DNA measured by time-resolved fluorescence, were in excellent agreement with the values obtained by picosecond laser flash photolysis. Similarly, fluorescence or absorption lifetime studies of the folate-depleted enzyme with and without photodimer suggest that FADH2, in its singlet excited state, transfers an electron to the dimer with 89% efficiency. The distance between FADH2 and the photodimer was calculated to be ca. 14 A.     

Localization of prolactin receptor in the dorsal and ventral skin of the frog (Rana ridibunda)

The dorsal and ventral skin in amphibians plays an important role in osmoregulation. Prolactin hormone is involved in regulation of amphibian skin functions, such as water and electrolyte balance. Therefore, amphibians may be useful as a model for determining the sites of the prolactin receptor. In this study, prolactin receptor was detected in frog dorsal and ventral skin using immunohistochemical staining method. Prolactin receptor immunoreactivity was localized in all epidermal layers except stratum corneum of dorsal skin epidermis, stratum germinativum layer of ventral skin epidermis, myoepithelial cells, secretory epithelium and secretory channel cells of granular glands in both skin regions. The mucous glands and secretory granules of granular glands did not show immunoreactivity for the prolactin receptor. According to our immunohistochemical results, the more widespread detection of prolactin receptor in dorsal skin epidermis indicates that prolactin is more effective in dorsal skin. Presence of prolactin receptors in epidermis points out its possible osmoregulatory effect. Moreover, detection of receptor immunoreactivity in various elements of poison glands in the dermis of both dorsal and ventral skin regions suggests that prolactin has a regulatory effect in gland functions.     

DNA photolyases: physical properties, action mechanism, and roles in dark repair

DNA photolyases catalyze the light-dependent repair of cis,syn-cyclobutane dipyrimidines (pyrimidine dimers). Although the phenomenon of enzymatic photoreactivation was first described 40 years ago and photolyases were the first enzymes shown unequivocally to effect DNA repair, it has only been in the last 8 years that sufficient quantities of the enzymes have been purified to permit detailed studies of their physical properties, identification of their intrinsic chromophores, and elucidation of the mechanisms of dimer recognition and photolysis. In addition several of the genes encoding these enzymes have now been cloned and sequenced. These studies have revealed remarkable functional and structural conservation among these evolutionarily ancient enzymes and have identified a new role for photolyases in dark-repair processes which has implications for the mechanism of nucleotide excision repair in both prokaryotes and eukaryotes.