Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.99.3 (PRE)
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Photolyase (photoreactivating enzyme) from the cyanobacterium Anacystis nidulans was crystallized by the hanging drop vapor diffusion procedure using ammonium sulfate as a precipitant. The pale-yellow crystals were grown to a size of 0.4 mm in length and 0.1 mm in diameter. They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 90.7 A and c = 135 A. Assuming that the asymmetric unit contains one molecule, the Vm value is calculated as 2.6 A3/dalton. The crystals are stable towards X-ray exposure and diffract beyond 2.5 A resolution.
J Mol Biol 1993 Sep 05
PMID:Crystallization and preliminary X-ray diffraction studies of photolyase (photoreactivating enzyme) from the cyanobacterium Anacystis nidulans. 837 84

Restriction enzymes, such as Eco RI, Hind III, etc., which have a potential pyrimidine dimer site in their recognition sequence, fail to cleave DNA if their recognition site is modified by the formation of pyrimidine dimers as a result of UV irradiation of DNA (J. E. Cleaver, J. Mol. Biol., 170 (1983) 305-317). We have made use of this functional property of restriction enzymes to develop a rapid and sensitive assay for DNA photolyases. UV-irradiated plasmid pBR322 DNA is only partially digested when incubated with a single site enzyme Hind III even at high concentration (20 units (micrograms DNA)-1). The amount of DNA not cleaved by Hind III is determined by agarose gel electrophoresis. The effect of UV irradiation is reversed by the photoreactivation of DNA. The decrease in the amount of Hind III-resistant DNA on treatment with photolyase gives a measure of the enzymatic activity of the photolyase preparation. The advantage of using non-radioactive DNA and the high speed and simplicity of this assay make it especially suitable for use in the purification of photolyases.
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PMID:An assay for DNA photolyases using non-radioactive DNA and restriction enzymes. 839 17

Using cells that overproduce DNA photolyase, we found that UV irradiation (3 J/m2) efficiently inactivates accumulation of methylthiogalactoside (TMG) when RexAB proteins of phage lambda are present. The effect requires both formation of photolyase-dimer-DNA (PDD) complexes and expression of the RexAB proteins. It is reversed completely by a flash of visible light if given immediately after UV and becomes irreversible after post-UV incubation for about 15 min. Inactivation is significant after only 5 min of post-UV incubation, is accompanied by a loss of previously accumulated TMG, and does not require de novo protein synthesis. Passive transport of O-nitrophenylgalactoside by inactivated cells is typical of energy-depleted membranes. We suggest that PDD complexes mimic a developmental intermediate of phage superinfection and stimulate formation of the RexB membrane channel recently proposed by others to explain classical "exclusion". This suggestion is supported by additional data showing an inactivation of colony-forming ability by exclusion stimulation and an inability of PDD complexes to inactivate accumulation of TMG if RexB is present in larger relative amounts than RexA (a detail characteristic of exclusion stimulated by phage superinfection).
Mol Gen Genet 1993 Sep
PMID:Photolyase-dimer-DNA complexes and exclusion stimulation in Escherichia coli: depolarization of the plasma membrane. 841 96

We have used in vitro DNA replication systems from human HeLa cells and monkey CV-1 cells to replicate a UV-damaged simian virus 40-based shuttle vector plasmid, pZ189. We found that replication of the plasmid was inhibited in a UV fluence-dependent manner, but even at UV fluences which caused damage to essentially all of the plasmid molecules some molecules became completely replicated. This replication was accompanied by an increase (up to 15-fold) in the frequency of mutations detected in the supF gene of the plasmid. These mutations were predominantly G:C-->A:T transitions similar to those observed in vivo. Treatment of the UV-irradiated plasmid DNA with Escherichia coli photolyase to reverse pyrimidine cyclobutane dimers (the predominant UV-induced photoproduct) before replication prevented the UV-induced inhibition of replication and reduced the frequency of mutations in supF to background levels. Therefore, the presence of pyrimidine cyclobutane dimers in the plasmid template appears to be responsible for both inhibition of replication and mutation induction. Further analysis of the replication of the UV-damaged plasmid revealed that closed circular replication products were sensitive to T4 endonuclease V (a pyrimidine cyclobutane dimer-specific endonuclease) and that this sensitivity was abolished by treatment of the replicated DNA with E. coli photolyase after replication but before T4 endonuclease treatment. These results demonstrate that these closed circular replication products contain pyrimidine cyclobutane dimers. Density labeling experiments revealed that the majority of plasmid DNA synthesized in vitro in the presence of bromodeoxyuridine triphosphate was hybrid density whether or not the plasmid was treated with UV radiation before replication; therefore, replication of UV-damaged templates appears to occur by the normal semiconservative mechanism. All of these data suggest that replication of UV-damaged templates occurs in vitro as it does in vivo and that this replication results in mutation fixation.
Mol Cell Biol 1993 Jan
PMID:Replication and mutagenesis of UV-damaged DNA templates in human and monkey cell extracts. 841 49

DNA photolyase from Escherichia coli (M(r) 54,000) consists of a polypeptide chain of 471 amino acids and the non-covalently bound cofactors methenyltetrahydrofolate (MTHF) and flavin adenine dinucleotide (FADH2). Two crystal forms of the enzyme were obtained; both have symmetry of space group P1. Form I has the unit cell dimensions a = 89.4 A, b = 97.3 A, c = 62.1 A, alpha = 108.3 degrees, beta = 97.4 degrees and gamma = 90.0 degrees. Diffraction from this form extends beyond 3 A resolution, but the crystals are radiation-sensitive and difficult to reproduce. Form II has the unit cell dimensions a = 62.6 A, b = 72.2 A, c = 58.5 A, alpha = 99.1 degrees, beta = 101.5 degrees and gamma = 72.0 degrees; most likely, the unit cell contains two molecules. High diffraction quality and reproducibility make form II suitable for structure analysis.
J Mol Biol 1993 Jun 20
PMID:Crystallization and preliminary crystallographic analysis of Escherichia coli DNA photolyase. 851 68

In order to better understand the biochemical mechanisms of DNA metabolism in chloroplasts, repair of UV induced plastome damage in vivo was determined by exposure of soybean suspension cells to UV light and subsequent quantitation of the damage remaining in nuclear and chloroplast encoded genes with time by quantitative polymerase chain reaction (QPCR). The kinetics of damage repair in the nuclear rbcS gene suggest that photoreactivation and dark mechanisms are active, while for the plastome encoded psbA gene only a light-dependent repair process was detected which is considerably slower than would be expected for photolyase-mediated photoreactivation.
Plant Mol Biol 1995 Dec
PMID:Repair mechanisms of UV-induced DNA damage in soybean chloroplasts. 861 23

The mammalian beta-like globin gene family has served as an important model system for analysis of tissue- and developmental state-specific gene regulation. Although the activities of a number of regulatory proteins have been implicated in the erythroid cell-specific transcription of globin genes, the mechanisms that restrict their expression to discrete stages of development are less well understood. We have previously identified a novel regulatory element (PRE II) upstream from the human embryonic beta-like globin gene (epsilon) that synergizes with other sequences to confer tissue- and stage-specific expression on a minimal epsilon-globin gene promoter in cultured embryonic erythroid cells. Binding of an erythroid nuclear protein (PRE II-binding factor [PRE-IIBF]) to the PRE II control element is required for promoter activation. Here we report on some of the biochemical properties of PREIIBF, including the characterization of its specificity and affinity for DNA. The embryonic and adult forms of PREIIBF recognize their cognate sequences with identical specificities, supporting our earlier conclusion that they are very similar proteins. PREIIBF binds DNA as a single polypeptide with an Mr of approximately 80,000 to 85,000 and introduces a bend into the target DNA molecule. These results suggest a mechanism by which PREIIBF may contribute to the regulation of the embryonic beta-like globin gene within the context of a complex locus.
Mol Cell Biol 1996 Mar
PMID:A DNA-bending protein interacts with an essential upstream regulatory element of the human embryonic beta-like globin gene. 862 84

In a comparative study of 17 pairs of 15 W fluorescent lamps intended for use in homes and purchased in local stores, we detect over 10-fold differences in UVB + UVC emissions between various lamps. This breakthrough of ultraviolet (UV) light is in part correlated with ability of lamps to kill DNA repair-defective recA-uvrB- Salmonella. Relative proficiency of lamps in eliciting photoreactivation of UV-induced DNA lesions also plays a prominent role in the relative rates of bacterial inactivation by emissions from different lamps. Lamps made in Chile, such as Philips brand lamps and one type of General Electric lamp, produce far less UVB + UVC and fail to kill recA-uvrB- bacteria. In contrast, all tested lamps manufactured in the USA, Hungary, and Japan exhibit readily observed deleterious biological effects. When an E. coli recA-uvrB-phr- (photolyase-negative) triple mutant is used for assay, lethal radiations are detected from all lamps, and single-hit exponential inactivation rates rather closely correlate to amount of directly measured UVB + UVC output of each pair of lamps. Although all lamps tested may meet international and United States standards for radiation safety, optimal practices in lamp manufacture are clearly capable of decreasing human exposure to indoor UV light.
Environ Mol Mutagen 1996
PMID:Breakthrough of ultraviolet light from various brands of fluorescent lamps: lethal effects on DNA repair-defective bacteria. 866 73

A cDNA from Arabidopsis thaliana similar to microbial photolyase genes, and designated AT-PHH1, was isolated using a photolyase-like cDNA from Sinapsis alba (SA-PHR1) as a probe. Multiple isolations yielded only PHH1 cDNAs, and a few blue-light-receptor CRY1 (HY4) cDNAs (also similar to microbial photolyase genes), suggesting the absence of any other highly similar Arabidopsis genes. The AT-PHH1 and SA-PHR1 cDNA sequences predict 89% identity at the protein level, except for an AT-PHH1 C-terminal extension (111 amino acids), also not seen in microbial photolyases. AT-PHH1 and CRY1 show less similarity (54% p4erein identity), including respective C-terminal extensions that are themselves mostly dissimilar. Analysis of fifteen AT-PHH1 genomic isolates reveals a single gene, with three introns in the coding sequence and one in the 5'-untranslated leader. Full-length AT-PHH1, and both AT-PHH1 and AT-PHH1 delta C-513 (truncated to be approximately the size of microbial photolyase genes) cDNAs, were overexpressed, respectively, in yeast and Escherichia coli mutants hypersensitive to ultraviolet light. The absence of significant effects on resistance suggests either that any putative AT-PHH1 DNA repair activity requires cofactors/chromophores not present in yeast or E. coli, or that AT-PHH1 encodes a blue-light/ultraviolet-A receptor rather than a DNA repair protein.
Mol Gen Genet 1996 Nov 27
PMID:PHH1, a novel gene from Arabidopsis thaliana that encodes a protein similar to plant blue-light photoreceptors and microbial photolyases. 900 12

Circular permutation analysis was used to determine the degree of DNA bending induced by binding of the glucocorticoid receptor (GR) DNA binding domain (DBD), the human progesterone receptor (PR) DBD, PR-A:A and PR-B:B homodimers, and PR-A:B heterodimers to the glucocorticoid response element/progesterone response element (GRE/PRE). The bending angles induced by the GR DBD and the PR DBD were approximately 28 degrees and 25 degrees, respectively. The PR-B:B and PR-A:A homodimers and the PR-A:B heterodimers all induced similar DNA bending angles of 72-77 degrees. The substantially greater DNA bend induced by full-length PR compared to the PR DBD indicates that sequences outside the classic zinc finger DNA binding domain may play an important role in the interaction of PR with the GRE/PRE. Because PR-A:A and PR-B:B homodimers and the PR-A:B heterodimers induce similar DNA bends, the different abilities of the PR-A and PR-B isoforms to activate transcription are not due to differences in their abilities to distort DNA structure.
J Steroid Biochem Mol Biol 1997 Jan
PMID:DNA bending is induced by binding of the glucocorticoid receptor DNA binding domain and progesterone receptors to their response element. 918 56


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