Pathways of electron transfer in Escherichia coli DNA photolyase: Trp306 to FADH.
We describe the results of a series of theoretical calculations of electron transfer pathways between Trp306 and *FADH. in the Escherichia coli DNA photolyase molecule, using the method of interatomic tunneling currents. It is found that there are two conformationally orthogonal tryptophans, Trp359 and Trp382, between donor and acceptor that play a crucial role in the pathways of the electron transfer process. The pathways depend vitally on the aromaticity of tryptophans and the flavin molecule. The results of this calculation suggest that the major pathway of the electron transfer is due to a set of overlapping orthogonal pi-rings, which starts from the donor Trp306, runs through Trp359 and Trp382, and finally reaches the flavin group of the acceptor complex, FADH. (+info
Rapid PCR test for discriminating between Candida albicans and Candida dubliniensis isolates using primers derived from the pH-regulated PHR1 and PHR2 genes of C. albicans.
The development of a satisfactory means to reliably distinguish between the two closely related species Candida albicans and Candida dubliniensis in the clinical mycology laboratory has proved difficult because these two species are phenotypically so similar. In this study, we have detected homologues of the pH-regulated C. albicans PHR1 and PHR2 genes in C. dubliniensis. Restriction fragment length polymorphism analysis suggests that there are significant sequence differences between the genes of the two species. In order to exploit this apparent difference, oligonucleotide primers based on the coding sequence of the C. albicans PHR1 structural gene were designed and used in PCR experiments. Use of these primers with C. albicans template DNA from 17 strains yielded a predicted 1.6-kb product, while C. dubliniensis template DNA from 19 strains yielded no product. We therefore propose that PCR using these primers is a rapid and reliable means of distinguishing the two germ tube- and chlamydospore-producing species C. albicans and C. dubliniensis. (+info
Rapid blue light regulation of a Trichoderma harzianum photolyase gene.
Photolyases and blue light receptors belong to a superfamily of flavoproteins that make use of blue and UVA light either to catalyze DNA repair or to control development. We have isolated a DNA photolyase gene (phr1) from Trichoderma harzianum, a common soil fungus that is of interest as a biocontrol agent against soil-borne plant pathogens and as a model for the study of light-dependent development. The sequence of phr1 is similar to other Class I Type I eukaryotic photolyase genes. Low fluences of blue light rapidly induced phr1 expression both in vegetative mycelia, which lack photoprotective pigments, and, to a greater extent, in conidiophores. Thus, visible light induces the development of pigmented, resistant spores as well as the expression of phr1, perhaps announcing in this way the imminent exposure to the more damaging short wavelengths of sunlight. Light induction of phr1 in non-sporulating mutants shows that a complete sporulation pathway is not required for photoregulation. The light requirements for photoinduction of phr1 were not altered in dimY photoperception mutants. This suggests that photoinduction of sporulation and of photolyase expression is distinct in their photoreceptor system or in the transduction of the blue light signal. (+info
Intraprotein electron transfer between tyrosine and tryptophan in DNA photolyase from Anacystis nidulans.
Light-induced electron transfer reactions leading to the fully reduced, catalytically competent state of the flavin adenine dinucleotide (FAD) cofactor have been studied by flash absorption spectroscopy in DNA photolyase from Anacystis nidulans. The protein, overproduced in Escherichia coli, was devoid of the antenna cofactor, and the FAD chromophore was present in the semireduced form, FADH., which is inactive for DNA repair. We show that after selective excitation of FADH. by a 7-ns laser flash, fully reduced FAD (FADH-) is formed in less than 500 ns by electron abstraction from a tryptophan residue. Subsequently, a tyrosine residue is oxidized by the tryptophanyl radical with t(1)/(2) = 50 microseconds. The amino acid radicals were identified by their characteristic absorption spectra, with maxima at 520 nm for Trp. and 410 nm for TyrO. The newly discovered electron transfer between tyrosine and tryptophan occurred for approximately 40% of the tryptophanyl radicals, whereas 60% decayed by charge recombination with FADH- (t(1)/(2) = 1 ms). The tyrosyl radical can also recombine with FADH- but at a much slower rate (t(1)/(2) = 76 ms) than Trp. In the presence of an external electron donor, however, TyrO. is rereduced efficiently in a bimolecular reaction that leaves FAD in the fully reduced state FADH-. These results show that electron transfer from tyrosine to Trp. is an essential step in the process leading to the active form of photolyase. They provide direct evidence that electron transfer between tyrosine and tryptophan occurs in a native biological reaction. (+info
Characterization of DNA recognition by the human UV-damaged DNA-binding protein.
The UV-damaged DNA-binding (UV-DDB) protein is the major factor that binds DNA containing damage caused by UV radiation in mammalian cells. We have investigated the DNA recognition by this protein in vitro, using synthetic oligonucleotide duplexes and the protein purified from a HeLa cell extract. When a 32P-labeled 30-mer duplex containing the (6-4) photoproduct at a single site was used as a probe, only a single complex was detected in an electrophoretic mobility shift assay. It was demonstrated by Western blotting that both of the subunits (p48 and p127) were present in this complex. Electrophoretic mobility shift assays using various duplexes showed that the UV-DDB protein formed a specific, high affinity complex with the duplex containing an abasic site analog, in addition to the (6-4) photoproduct. By circular permutation analyses, these DNA duplexes were found to be bent at angles of 54 degrees and 57 degrees in the complexes with this protein. From the previously reported NMR studies and the fluorescence resonance energy transfer experiments in the present study, it can be concluded that the UV-DDB protein binds DNA that can be bent easily at the above angle. (+info
Class II DNA photolyase from Arabidopsis thaliana contains FAD as a cofactor.
The major UV-B photoproduct in DNA is the cyclobutane pyrimidine dimer (CPD). CPD-photolyases repair this DNA damage by a light-driven electron transfer. The chromophores of the class II CPD-photolyase from Arabidopsis thaliana, which was cloned recently [Taylor, R., Tobin, A. & Bray, C. (1996) Plant Physiol. 112, 862; Ahmad, M., Jarillo, J.A., Klimczak, L.J., Landry, L.G., Peng, T., Last, R.L. & Cashmore, A.R. (1997) Plant Cell 9, 199-207], have not been characterized so far. Here we report on the overexpression of the Arabidopsis CPD photolyase in Escherichia coli as a 6 x His-tag fusion protein, its purification and the analysis of the chromophore composition and enzymatic activity. Like class I photolyase, the Arabidopsis enzyme contains FAD but a second chromophore was not detectable. Despite the lack of a second chromophore the purified enzyme has photoreactivating activity. (+info
DNA bending by photolyase in specific and non-specific complexes studied by atomic force microscopy.
Specific and non-specific complexes of DNA and photolyase are visualised by atomic force microscopy. As a substrate for photolyase a 1150 bp DNA restriction fragment was UV-irradiated to produce damaged sites at random positions. Comparison with a 735 bp undamaged DNA fragment made it possible to separate populations of specific and non-specific photolyase complexes on the 1150 bp fragment, relieving the need for highly defined substrates. Thus it was possible to compare DNA bending for specific and non-specific interactions. Non-specific complexes show no significant bending but increased rigidity compared to naked DNA, whereas specific complexes show DNA bending of on average 36 degrees and higher flexibility. A model obtained by docking shows that photolyase can accommodate a 36 degrees bent DNA in the vicinity of the active site. (+info
RPH1 and GIS1 are damage-responsive repressors of PHR1.
The Saccharomyces cerevisiae DNA repair gene PHR1 encodes a photolyase that catalyzes the light-dependent repair of pyrimidine dimers. PHR1 expression is induced at the level of transcription by a variety of DNA-damaging agents. The primary regulator of the PHR1 damage response is a 39-bp sequence called URS(PHR1) which is the binding site for a protein(s) that constitutes the damage-responsive repressor PRP. In this communication, we report the identification of two proteins, Rph1p and Gis1p, that regulate PHR1 expression through URS(PHR1). Both proteins contain two putative zinc fingers that are identical throughout the DNA binding region, and deletion of both RPH1 and GIS1 is required to fully derepress PHR1 in the absence of damage. Derepression of PHR1 increases the rate and extent of photoreactivation in vivo, demonstrating that the damage response of PHR1 enhances cellular repair capacity. In vitro footprinting and binding competition studies indicate that the sequence AG(4) (C(4)T) within URS(PHR1) is the binding site for Rph1p and Gis1p and suggests that at least one additional DNA binding component is present in the PRP complex. (+info