DNA photolyase homologs are the major UV resistance factors in the cyanobacterium Synechocystis sp. PCC 6803

In this study, the unicellular photosynthetic cyanobacterium Synechocystis sp. PCC 6803 was used as a model phototroph to study the contribution of enzymatic photoreactivation to the overall protection against UV irradiation. We have isolated genes encoding two DNA photolyase homologs, phrA and phrB, from Synechocystis 6803. phrA encodes an 8-hydroxy-5-deazariboflavin (HDF) type, Class I DNA photolyase. By complementing a photolyase-deficient mutant strain of Escherichia coli, we demonstrated that PhrA is a DNA photolyase. Analysis of a phrA knockout mutant strain suggested that this gene is responsible for the majority of the observed UV resistance in Synechocystis 6803. Similar studies on phrB demonstrated that it also contributes to photoreactivation, but to a much lesser degree. Based on these findings, we conclude that enzymatic photoreactivation is the primary process used for repairing UV-induced damage in Synechocystis 6803.     

Reverse gyrase recruitment to DNA after UV light irradiation in Sulfolobus solfataricus

Induction of DNA damage triggers a complex biological response concerning not only repair systems but also virtually every cell function. DNA topoisomerases regulate the level of DNA supercoiling in all DNA transactions. Reverse gyrase is a peculiar DNA topoisomerase, specific to hyperthermophilic microorganisms, which contains a helicase and a topoisomerase IA domain that has the unique ability to introduce positive supercoiling into DNA molecules. We show here that reverse gyrase of the archaean Sulfolobus solfataricus is mobilized to DNA in vivo after UV irradiation. The enzyme, either purified or in cell extracts, forms stable covalent complexes with UV-damaged DNA in vitro. We also show that the reverse gyrase translocation to DNA in vivo and the stabilization of covalent complexes in vitro are specific effects of UV light irradiation and do not occur with the intercalating agent actinomycin D. Our results suggest that reverse gyrase might participate, directly or indirectly, in the cell response to UV light-induced DNA damage. This is the first direct evidence of the recruitment of a topoisomerase IA enzyme to DNA after the induction of DNA damage. The interaction between helicase and topoisomerase activities has been previously proposed to facilitate aspects of DNA replication or recombination in both Bacteria and Eukarya. Our results suggest a general role of the association of such activities in maintaining genome integrity and a mutual effect of DNA topology and repair.     

Identification of a DNA supercoiling activity in Saccharomyces cerevisiae.

A relaxed plasmid DNA is shown to become positively supercoiled in cell extracts from top1 strains of Saccharomyces cerevisiae. This positive supercoiling activity is dependent on the presence of bacterial DNA topoisomerase I and ATP (or dATP), and the positive supercoils generated in this reaction are not constrained by protein(s). Non-hydrolyzable ATP analogs cannot substitute for ATP in this supercoiling reaction, and the supercoiling activity is not due to RNA synthesis. The presence of an ARS sequence in the DNA does not alter the activity. Furthermore, this activity is equally active against UV irradiated or intact DNA. Extracts prepared from rad50 and rad52 mutant cells exhibited the same activity. Partial purification of this activity suggests that a protein factor with a native molecular weight of approximately 150 kDa is primarily responsible for the activity. The possibility that this supercoiling activity may be due to tracking of a protein along the intact duplex DNA is discussed.     

The essential DNA polymerases delta and epsilon are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae.

We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae. By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases delta and epsilon showed accumulation of single-strand breaks indicating defective repair. Mutations in other DNA polymerase genes exhibited no defects in DNA repair. Thus, the data obtained suggest that DNA polymerases delta and epsilon are both necessary for DNA replication and for repair of lesions caused by UV irradiation. The results are discussed in the light of current concepts concerning the specificity of DNA polymerases in DNA repair.     

Photocontrol of kinesin ATPase activity using an azobenzene derivative

Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-forms in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Here, we introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the functional region of kinesin to reversibly regulate the ATPase activity of kinesin by photoirradiation. We prepared five kinesin motor domain mutants, A247C, L249C, A252C, G272C and S275C, which contained a single reactive cysteine residue in loops L11 and L12. These loops are considered to be key regions for the functioning of kinesin as a motor protein. PAM was stoichiometrically incorporated into the cysteine residues in the loops of the mutants. The PAM-modified S275C mutant exhibited reversible alterations in ATPase activity accompanied by cis-trans isomerization upon UV and VIS light irradiation. The ATPase activity exhibited by the cis-isomer of the PAM bound to the mutant was two times higher than that of the trans-isomer. Further, the PAM-modified L249C mutant exhibited reversible alterations in ATPase activity on UV-VIS light irradiation but exhibited the opposite effect on UV and VIS light irradiation. Using a photochromic azobenzene derivative, we have demonstrated that the ATPase activity of the motor protein kinesin is photoregulated.     

Visible laser and UV-A radiation impact on a PNP degrading Moraxella strain and its rpoS mutant

The role of stationary phase sigma factor gene (rpoS) in the stress response of Moraxella strain when exposed to radiation was determined by comparing the stress responses of the wild-type (WT) and its rpoS knockout (KO) mutant. The rpoS was turned on by starving the WT cultures for 24 h in minimal salt medium. Under non-starved condition, both WT and KO planktonic Moraxella cells showed an increase in mortality with the increase in duration of irradiation. In the planktonic non-starved Moraxella, for the power intensity tested, UV radiation caused a substantially higher mortality rate than did by the visible laser light (the mortality rate observed for 15-min laser radiation was 53.4 +/- 10.5 and 48.7 +/- 8.9 for WT and KO, respectively, and 97.6 +/- 0 and 98.5 +/- 0 for 25 s of UV irradiation in WT and KO, respectively). However, the mortality rate decreased significantly in the starved WT when exposed to these two radiations. In comparison, rpoS protected the WT against the visible laser light more effectively than it did for the UV radiation. The WT and KO strains of Moraxella formed distinctly different types of biofilms on stainless steel coupons. The KO strain formed a denser biofilm than did the WT. Visible laser light removed biofilms from the surfaces more effectively than did the UV. This was true when comparing the mortality of bacteria in the biofilms as well. The inability of UV radiation to penetrate biofilms due to greater rates of surface absorption is considered to be the major reason for the weaker removal of biofilms in comparison to that of the visible laser light. This result suggests that high power visible laser light might be an effective tool for the removal of biofilms.     

Photoswitching of ligand association with a photoresponsive polymer-protein conjugate

Light-regulated molecular switches that reversibly control biomolecular function could provide new opportunities for controlling activity in diagnostics, affinity separations, bioprocessing, therapeutics, and bioelectronics applications. Here we show that site-specific conjugation of light-responsive polymers near the biotin-binding pocket of streptavidin provides control of ligand binding affinity in response to UV and visible light irradiation. Two different light-responsive polymers were utilized that display opposite photoresponsive solubility changes under UV or visible (vis) light irradiation in aqueous solutions. At 40 degrees C, the N,N-dimethylacrylamide (DMA)-co-4-phenylazophenyl acrylate (AZAA) copolymer (DMAA) was soluble under UV irradiation and precipitated under visible light, while the DMA-co-N-4-phenylazophenyl acrylamide (AZAAm) copolymer (DMAAm) was soluble under visible irradiation and precipitated under UV light. Both polymers were synthesized with a vinyl sulfone terminus and conjugated to the Glu116Cys (E116C) streptavidin mutant via thiol coupling. The DMAA-streptavidin conjugate bound biotin efficiently when the polymer was in the soluble state under UV irradiation, but under visible irradiation, the polymer collapsed and blocked free biotin association. Furthermore, if biotin was allowed to bind when the polymer was in the soluble state under UV irradiation, then when the polymer was collapsed by visible light, the streptavidin released the bound biotin. The DMAAm-streptavidin conjugate showed the opposite response, with association of biotin allowed under visible light irradiation and blocked under UV irradiation. The photoresponses of the streptavidin conjugates thus correspond to the original photoresponsive phase transition properties of the polymer switches triggered by the cis-trans isomerization of the diazo chromophores.     

Stimulation of DNA repair and increased light output in response to UV irradiation in Escherichia coli expressing lux genes.

It has previously been suggested that the evolutionary drive of bacterial bioluminescence is a mechanism of DNA repair. By assessing the UV sensitivity of Escherichia coli, it is shown that the survival of UV-irradiated E. coli constitutively expressing luxABCDE in the dark is significantly better than either a strain with no lux gene expression or the same strain expressing only luciferase (luxAB) genes. This shows that UV resistance is dependent on light output, and not merely on luciferase production. Also, bacterial survival was found to be dependent on the conditions following UV irradiation, as bioluminescence-mediated repair was not as efficient as repair in visible light. Moreover, photon emission revealed a dose-dependent increase in light output per cell after UV exposure, suggesting that increased lux gene expression correlates with UV-induced DNA damage. This phenomenon has been previously documented in organisms where the lux genes are under their natural luxR regulation but has not previously been demonstrated under the regulation of a constitutive promoter.     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.     

The effect of nutritional state on photoreversal of ultraviolet injuries in Didinium nasutum

1. The effect of the nutritional state of Didinium nasutum on its resistance to short ultraviolet (UV) radiation (2654 A) and its recovery from the injury following illumination with visible light (4350 A, blue) was studied. 2. The resistance of a didinium to UV is considerably increased by feeding it a paramecium 15 to 60 minutes before exposure to UV. If fed just before exposure to UV, the resistance is less than that of an unfed control. 3. Photoreversal is only slightly greater in didinia fed after irradiation with UV but before exposure to visible light as compared to those fed after exposure to visible light. 4. Irradiated paramecia are eaten by didinia, provided they have not started to cytolyze. Didinia fed on irradiated paramecia divide at about the same rate as controls or slightly faster. 5. The available stock of Didinium declines in vigor with lapse of time after excystment, as measured by the time required for division. The sensitivity of Didinium to UV did not change essentially during the 5 month period over which tests were made. 6. The theoretical implications of the results are considered.     

Mechanism of differential staining of BUdR-substituted Vicia faba chromosomes.

Chromosomes of the broad bean Vicia faba were isolated and air-dried on slides after incorporation of BUdR into DNA (BUdR substitution) for two rounds of replication. Then the preparations were embedded in a buffer solution containing trypsin as well as fluorescence dye (acridine orange or Hoechst 33258). We observed chromosomes with a fluorescence microscope at various times after embedding. After about 15 min one sister chromatid of some of the metaphase chromosomes showed enhanced darkening and disintegration within 1–4 min (melting effect) during observation. We suppose that fragmentation of BUdR-substituted DNA by the acridine orange-visible light system in acridine orange staining and by irradiation with wavelengths around the transition from UV to visible light in Hoechst 33258 staining is responsible for this phenomenon. The disintegration of one sister chromatid in BUdR-substituted chromosomes can also be produced by UV irradiation during trypsin treatment when fluorescence dyes are not present.     

Excision Repair Characteristics of recB−res− and uvrC− Strains of Escherichia coli

An Escherichia coli strain carrying the recB21 and res-1 mutations showed an abnormally low level of colony-forming ability although it grew essentially normally in liquid medium. The recB21 res-1 strain showed little, if any, of the ultraviolet (UV)-induced deoxyribonucleic acid (DNA) breakdown characteristic of the res-1 mutant. Nevertheless, the double mutant was far more sensitive to UV than either the res-1 or the recB21 strain. When compared with a wild-type strain, the rate of release of dimers from UV-irradiated DNA was very slow in the recB21 res-1, but normal in the res-1 recB(+) or recB21 res(+) mutants. However, the ratio of dimer-to-thymine released into the acid-soluble fraction was three times higher than the wild type in recB21 res(+) and recB21 res-1 and only one-tenth as high as the wild type in res-1 rec(+). Alkaline sucrose gradient centrifugation revealed occurrence of single-strand incision of UV-irradiated DNA and the restitution of nicked DNA at a similar rate in the recB21 res-1 and recB21 res(+) strains. Mutants uvrC(-) showed increased amounts of nicks in their DNA with increasing incubation time after UV irradiation, although no detectable amounts of dimers were excised from UV-irradiated DNA. From these results, it is concluded that the increased sensitivity of the res-1 strain to UV light is due to a reduced ability to excise dimers from UV-irradiated DNA and that the high rate of UV-induced breakdown of DNA is not the primary cause. A possible role of uvrC gene in the excision repair is discussed.     

Pleiotropic effects of heterozygosity at the mating-type locus of the yeast Saccharomyces cerevisiae on repair, recombination and transformation

Sexual (MAT a/) and sexual (MAT a/a) strains of the yeast Saccharomyces cerevisiae, which are completely isogenic except at the MAT locus, were compared in their response to ultraviolet radiation. The effects of UV on survival, mitotic intragenic recombination, photoreactivation, and transformation efficiency with UV-irradiated plasmid DNA were examined. The sexual strain had enhanced survival and higher rates of mitotic intragenic recombination compared with the asexual strain. Exposure to visible light subsequent to irradiation increased the survival of both sexual and asexual strains, and decreased their rates of mitotic intragenic recombination. Similar results were obtained by Haladus and Zuk (1980) in their examination of sexual strains homozygous for rad6-1, and wild-type sexuals.

Our sexual strain was also consistently more proficient at transforming plasmid DNA, whether that DNA had been irradiated or not. When pre-irradiated with 25 J/m² of UV, MAT a/ cells transformed more efficiently than MAT a/a cells. When subsequently exposed to light, the ability of these pre-irradiated cells to transform decreased for both strains with increasing irradiation of the plasmid. A smaller decrease in transformation efficiency occurred when cells of both strains were kept in the dark.

When pre-irradiated with 100 J/m², the MAT a/ cells showed a 2-fold increase in their transformation efficiency of both irradiated and unirradiated plasmids by up to 2-fold, a phenomenon not seen in the MAT a/a cells even when pre-irradiated with much higher doses of UV. This increase in transformation efficiency was not, however, seen in the MAT a/ cells when they were exposed to visible light after UV irradiation. These results suggest that cells with the MAT a genotype have a UV-inducible system that increases the efficiency of transformation in the absence of visible light. This increase in transformation is not an induced increase in the repair of plasmid DNA, but rather an increase in the ability of pre-irradiated MAT a/ cells to take up exogenous DNA. MAT a/a cells do not appear to have a similarity inducible system. To the best of our knowledge, this phenomenon has not been previously reported.     

RecO is essential for DNA damage repair in Deinococcus radiodurans

Here we present direct evidence for the vital role of RecO in Deinococcus radiodurans's radioresistance. A recO null mutant was constructed using a deletion replacement method. The mutant exhibited a growth defect and extreme sensitivity to irradiation with gamma rays and UV light. These results suggest that DNA repair in this organism occurs mainly via the RecF pathway.     

Fibromodulin gene transcription is induced by ultraviolet irradiation, and its regulation is impaired in senescent human fibroblasts

Cells undergoing replicative senescence display an altered pattern of gene expression. Senescent fibroblasts show significant changes in the expression of mRNAs encoding extracellular matrix-remodeling proteins; among these mRNAs, the mRNA encoding fibromodulin is highly decreased in these cells. To understand the molecular basis of this phenomenon, we explored the regulatory mechanisms of the human fibromodulin gene. We found that fibromodulin gene promoter contains a cis-element, crucial for its basal expression, that forms a DNA-protein complex when exposed to nuclear extracts from exponentially growing human fibroblasts and not to extracts from cells undergoing senescence by repeated in vitro passages or by mild oxidative stress. The purification of this complex showed that it contains the damage-specific DNA-binding protein DDB-1. The latter is known to be induced by UV irradiation; therefore we checked whether fibromodulin gene promoter is regulated upon the exposure of the cells to UV rays. The results showed that, in exponentially growing fibroblasts, the promoter efficiency is increased by UV irradiation and the DDB-1-containing complex is robustly enriched in cells exposed to UV light. Accordingly, in these experimental conditions the endogenous fibromodulin mRNA accumulates to very high levels. On the contrary, senescent cells did not show any activation of the fibromodulin gene promoter, any induction of the DDB-1-containing complex, or any accumulation of fibromodulin mRNA. These phenomena are accompanied in senescent cells by a decrease of the UV-damaged DNA binding activity.     

Temperature effects on Agrobacterium phytochrome Agp1

Phytochromes are widely distributed biliprotein photoreceptors with a conserved N-terminal chromophore-binding domain. Most phytochromes bear a light-regulated C-terminal His kinase or His kinase-like region. We investigated the effects of light and temperature on the His kinase activity of the phytochrome Agp1 from Agrobacterium tumefaciens. As in earlier studies, the phosphorylation activity of the holoprotein after far-red irradiation (where the red-light absorbing Pr form dominates) was stronger than that of the holoprotein after red irradiation (where the far red-absorbing Pfr form dominates). Phosphorylation activities of the apoprotein, far red-irradiated holoprotein, and red-irradiated holoprotein decreased when the temperature increased from 25 °C to 35 °C; at 40 °C, almost no kinase activity was detected. The activity of a holoprotein sample incubated at 40 °C was nearly completely restored when the temperature returned to 25 °C. UV/visible spectroscopy indicated that the protein was not denatured up to 45 °C. At 50 °C, however, Pfr denatured faster than the dark-adapted sample containing the Pr form of Agp1. The Pr visible spectrum was unaffected by temperatures of 20-45 °C, whereas irradiated samples exhibited a clear temperature effect in the 30-40 °C range in which prolonged irradiation resulted in the photoconversion of Pfr into a new spectral species termed Prx. Pfr to Prx photoconversion was dependent on the His-kinase module of Agp1; normal photoconversion occurred at 40 °C in the mutant Agp1-M15, which lacks the C-terminal His-kinase module, and in a domain-swap mutant in which the His-kinase module of Agp1 is replaced by the His-kinase/response regulator module of the other A. tumefaciens phytochrome, Agp2. The temperature-dependent kinase activity and spectral properties in the physiological temperature range suggest that Agp1 serves as an integrated light and temperature sensor in A. tumefaciens.