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At least 19 recordsLinked to original sources
PubMed1999

Human DNA repair systems: an overview.

DNA repair systems act to maintain genome integrity in the face of replication errors, environmental insults, and the cumulative effects of age. More than 70 human genes directly involved in the five major pathways of DNA repair have been described, including chromosomal location and cDNA sequence. However, a great deal of information as to the precise functions of these genes and their role in human health is still lacking. Hence, we summarize what is known about these genes and their contra part in bacterial, yeast, and rodent systems and discuss their involvement in human disease. While some associations are already well understood, it is clear that additional diseases will be found which are linked to DNA repair defects or deficiencies.

Animals↗
PubMed1998

Alternative repair pathways for UV-induced DNA damage.

Ultraviolet light (UV) is thought to have had a major impact on the early evolution of life. UV is absorbed by nucleic acids and produces several types of DNA damage, which interfere with DNA replication and transcription. This damage can result in mutagenesis and cell killing. Several mechanisms for repairing UV-induced DNA damage have been identified. Besides the widely distributed nucleotide excision repair, two alternative repair mechanisms for specific lesions in UV-damaged DNA are known, involving photolyases and DNA glycosylases. Recently, a novel endonuclease for UV-induced DNA damage was identified that initiates an excision repair pathway completely different from previously established repair mechanisms. The finding of this "alternative excision repair" suggests the presence of a new category of DNA repair, initiated by single-strand breaks in DNA. Homologues of the UVDE enzyme have been found in eukaryotic microorganisms, as well as in bacteria, indicating that the enzyme originated early in evolution, and suggesting the existence of multirepair systems for UV-induced DNA damage during early evolution.

DNA↗
PubMed2000

A comparative repair study of thymine- and uracil-photodimers with model compounds and a photolyase repair enzyme.

Cyclobutane uridine and thymidine dimers with cis-syn-structure are DNA lesions, which are efficiently repaired in many species by DNA photolyases. The essential step of the repair reaction is a light driven electron transfer from a reduced FAD cofactor (FADH ) to the dimer lesion, which splits spontaneously into the monomers. Repair studies with UV-light damaged DNA revealed significant rate differences for the various dimer lesions. In particular the effect of the almost eclipsed positioned methyl groups at the thymidine cyclobutane dimer moiety on the splitting rates is unknown. In order to investigate the cleavage vulnerability of thymine and uracil cyclobutane photodimers outside the protein environment, two model compounds, containing a thymine or a uracil dimer and a covalently connected flavin, were prepared and comparatively investigated. Cleavage investigations under internal competition conditions revealed, in contrast to all previous findings, faster repair of the sterically less encumbered uracil dimer. Stereoelectronic effects are offered as a possible explanation. Ab initio calculations and X-ray crystal structure data reveal a different cyclobutane ring pucker of the uracil dimer, which leads to a better overlap of the pi*-C(4)-O(4)-orbital with the sigma*-C(5)-C(5')-orbital. Enzymatic studies with a DNA photolyase (A. nidulans) and oligonucleotides, which contain either a uridine or a thymidine dimer analogue, showed comparable repair efficiencies for both dimer lesions. Under internal competition conditions significantly faster repair of uridine dimers is observed.

Aspergillus nidulans↗
PubMed2002

Cleavable substrate containing molecular beacons for the quantification of DNA-photolyase activity.

In order to gain deeper insight into the function and interplay of proteins in cells it is essential to develop methods that allow the profiling of protein function in real time, in solution, in cells, and in cell organelles. Here we report the development of a U-type oligonucleotide (molecular beacon) that contains a fluorophore and a quencher at the tips, and in addition a substrate analogue in the loop structure. This substrate analogue induces a hairpin cleavage in response to enzyme action, which is translated into a fluorescence signal. The molecular beacon developed here was used to characterize DNA-photolyase activity. These enzymes represent a challenge for analytical methods because of their low abundance in cells. The molecular beacon made it possible to measure the activity of purified class I and class II photolyases. Photolyase activity was even detectable in crude cell extracts.

DNA Repair↗
PubMed1993

Substituent effects on the puckering mode of the cyclobutane ring and the glycosyl bond of cis-syn photodimers.

The cyclobutane ring (CB) puckering of a cis-syn DNA photodimer (cis-syn d-T[p]T) differs from that of a cis-syn RNA photodimer (cis-syn r-U[p]U) [J.-K. Kim and J.L. Alderfer (1992) Journal of Biomolecular Structure and Dynamics, Vol. 9, p. 1705]. In cis-syn d-T[p]T, interconversion of the CB ring between CB+ and CB- is observed, while in cis-syn r-U[p]U only CB- is observed. In the CB+ conformation, the two thymine rings of the dimer are twisted in a right-handed fashion, as are the bases in B-form DNA. In case of CB- they are twisted in a left-handed fashion. The C5 (base) and/or C2' (sugar) substituents apparently affect the CB ring flexibility in cis-syn d-T[p]T and cis-syn r-U[p]U. To study the effects of the C5 substituent on CB ring flexibility, two-dimensional nuclear Overhauser effect (NOE) and 31P-nmr experiments were performed on cis-syn d-T[p]U, cis-syn d-U[p]T, and cis-syn d-U[p]U photodimers to investigate the CB puckering mode and overall molecular conformation and dynamics. The NOE results indicate the 5-methyl group in the photodimer induces conformational flexibility of the CB ring. In cis-syn d-T[p]U and cis-syn d-U[p]T, both CB+ and CB- puckering modes are observed. This indicates interconversion between two modes takes place as observed in cis-syn d-T[p]T. In the case of cis-syn d-U[p]U, only the puckering CB- mode is observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclobutanes↗
PubMed2004

Preliminary characterization of light harvesting in E. coli DNA photolyase.

E. coli DNA photolyase is a monomeric light-harvesting enzyme that utilizes a methenyltetrahydrofolate (MTHF) antenna cofactor to harvest light energy for the repair of thymine dimers in DNA. For this purpose, the enzyme evolved to bind the cofactor and red-shift its absorption maximum by 25 nm. Using the crystal structure as a guide, we mutated each protein residue that contacts the cofactor in an effort to identify the interactions responsible for this selective stabilization of the cofactor's excited state. Hydrogen bonding, packing, and electrostatic interactions were examined. Remarkably, a single residue, Glu109, appears to play an important, if not exclusive, role in inducing the observed red-shift. Thus, this protein, the simplest light-harvesting system known, appears to have evolved a remarkably simple mechanism to tune the photophysical properties of the antenna cofactor appropriately for biological function.

Deoxyribodipyrimidine Photo-Lyase↗
PubMed2006

Natural and non-natural antenna chromophores in the DNA photolyase from Thermus thermophilus.

X-ray crystallographic and functional analysis of the class I DNA photolyase from Thermus thermophilus revealed the binding of flavin mononucleotide (FMN) as an antenna chromophore. The binding mode of FMN closely coincides with the binding of a deazaflavin-like chromophore in the related class I DNA photolyase from Anacystis nidulans. Compared to the R46E mutant, which lacks a conserved arginine in the binding site for the antenna chromophore, the FMN-comprising holophotolyase exhibits an eightfold higher activity at 450 nm. The facile incorporation of the flavin cofactors 8-hydroxy-deazariboflavin and 8-iodo-8-demethyl-riboflavin into the binding site for the antenna chromophore paves the way for wavelength-tuning of the activity spectra of DNA photolyases by using synthetic flavins.

Binding Sites↗
PubMed1995

Centripetal transport of microtubules in motile cells.

The study of microtubule (MT) dynamics in cells has largely been restricted to events occurring over relatively short periods in nonmotile or stationary cells in culture. By using the antioxidant, Oxyrase, we have reduced the sensitivity of fluorescent MTs to photodamage and this has allowed us to image fluorescent MTs with good temporal resolution over much longer periods of time. We have used our enhanced imaging capabilities to examine MT dynamics in fibroblasts moving directionally into a wound. We found that MTs in these cells exhibited dynamic instability similar to that reported for other cells. More interestingly, we found a novel dynamic behavior of the MTs in which entire MTs were moved inward from the leading edge toward the cell nucleus. This centripetal transport (CT) of MTs only occurred to those MTs that were oriented with their long axis parallel to the leading edge; radially oriented MTs were not transported centripetally. Both small bundles of MTs and individual MTs were observed to undergo CT at a rate of 0.63 +/- 0.37 micron/min. This rate was similar to the rate of CT of latex beads applied to the cell surface and of endogenous pinocytotic vesicles in the cytoplasm. When we imaged both MTs and pinocytotic vesicles, we found that the pinocytotic vesicles were ensheathed by a small group of parallel MTs that moved centripetally in concert with the vesicles. Conversely, we found many instances of MTs moving centripetally without associated vesicles. When cells were treated with nocodazole to depolymerize MTs rapidly, the rate of pinocytotic vesicle CT was inhibited by 75%. This suggests that centripetal transport of MTs may be involved in the movement of pinocytotic vesicles in cells. In conclusion, our results show that MTs in motile cells are redistributed by a novel mechanism, CT, that does not require changes in polymer length. The centripetally transported MTs may play a role in transporting pinocytotic vesicles in the cell.

Amidines↗
PubMed2005

Probing the N(5)-H bond of the isoalloxazine moiety of flavin radicals by X- and W-band pulsed electron-nuclear double resonance.

An X- (9.7 GHz and W-band (94 GHz) pulsed electron-nuclear double resonance (ENDOR) study of the flavin cofactor of Escherichia coli DNA photolyase in its neutral radical form is presented. Through proton and deuteron ENDOR measurements at T = 80 K, we detect and characterize the full anisotropy of the hyperfine coupling (hfc) tensor of the proton or deuteron bound to N(5) of the isoalloxazine ring. Scaling of the anisotropic proton hfc components by multiplication with the quotient of the magnetogyric ratio of a deuteron and a proton, chiD/chiH, reveals subtle differences compared to the respective deuteron couplings obtained by 95-GHz deuterium ENDOR spectroscopy on an H-->D buffer-exchanged sample. These differences can be attributed to the different lengths of N(5)-H and N(5)-D bonds arising from the different masses of protons and deuterons. From the R(-3) dependence of the dipolar hyperfine splitting, we estimated that the N(5)-D bond is about 2.5% shorter than the respective N(5)-H bond. That such subtle bond-length differences can be resolved by pulsed ENDOR spectroscopy suggests that this method may be favorably used to probe the geometry of hydrogen bonds between the H(5) of the paramagnetic flavin and the protein backbone. Such information is only obtained with difficulty by other types of spectroscopy.

Anisotropy↗
PubMed1993

DNA damage, photorepair, and survival in fish and human cells exposed to UV radiation.

The effect of various wavelengths of UVB radiation on the induction of cyclobutane pyrimidine dimers in fish cells and human fibroblasts and the repair of these lesions were studied using an UV-endonuclease to measure dimers (endonuclease sensitive sites) by sedimentation of radioactive DNA, by gel electrophoresis of unlabeled DNA, and by cell survival. The data show that fish cells have an efficient photoreactivation system at wavelength > 304 nm that reverses cytotoxicity and dimer formation after exposure to filtered sunlamp irradiation of a shorter wavelength (lambda > 290 nm). Shorter wavelengths in UVB (> 304 nm) are more effective in photoreversal than longer ones (> 320 nm). As a consequence, 50-85% of dimers induced by these wavelengths in fish are photoreactivated while they are being formed. A major cytotoxicological lesion is the cyclobutane pyrimidine dimers. Cultured human fibroblasts do not possess such a repair system. These results indicate that sunlamp irradiation has wavelengths that both damage and repair DNA.

Animals↗
PubMed1994

A new class of DNA photolyases present in various organisms including aplacental mammals.

DNA photolyase specifically repairs UV light-induced cyclobutane-type pyrimidine dimers in DNA through a light-dependent reaction mechanism. We have obtained photolyase genes from Drosophila melanogaster (fruit fly), Oryzias latipes (killifish) and the marsupial Potorous tridactylis (rat kangaroo), the first photolyase gene cloned from a mammalian species. The deduced amino acid sequences of these higher eukaryote genes show only limited homology with microbial photolyase genes. Together with the previously cloned Carassius auratus (goldfish) gene they form a separate group of photolyase genes. A new classification for photolyases comprising two distantly related groups is proposed. For functional analysis P.tridactylis photolyase was expressed and purified as glutathione S-transferase fusion protein from Escherichia coli cells. The biologically active protein contained FAD as light-absorbing cofactor, a property in common with the microbial class photolyases. Furthermore, we found in the archaebacterium Methanobacterium thermoautotrophicum a gene similar to the higher eukaryote photolyase genes, but we could not obtain evidence for the presence of a homologous gene in the human genome. Our results suggest a divergence of photolyase genes in early evolution.

Amino Acid Sequence↗
PubMed2002

Monophyly of class I aminoacyl tRNA synthetase, USPA, ETFP, photolyase, and PP-ATPase nucleotide-binding domains: implications for protein evolution in the RNA.

Protein sequence and structure comparisons show that the catalytic domains of Class I aminoacyl-tRNA synthetases, a related family of nucleotidyltransferases involved primarily in coenzyme biosynthesis, nucleotide-binding domains related to the UspA protein (USPA domains), photolyases, electron transport flavoproteins, and PP-loop-containing ATPases together comprise a distinct class of alpha/beta domains designated the HUP domain after HIGH-signature proteins, UspA, and PP-ATPase. Several lines of evidence are presented to support the monophyly of the HUP domains, to the exclusion of other three-layered alpha/beta folds with the generic "Rossmann-like" topology. Cladistic analysis, with patterns of structural and sequence similarity used as discrete characters, identified three major evolutionary lineages within the HUP domain class: the PP-ATPases; the HIGH superfamily, which includes class I aaRS and related nucleotidyltransferases containing the HIGH signature in their nucleotide-binding loop; and a previously unrecognized USPA-like group, which includes USPA domains, electron transport flavoproteins, and photolyases. Examination of the patterns of phyletic distribution of distinct families within these three major lineages suggests that the Last Universal Common Ancestor of all modern life forms encoded 15-18 distinct alpha/beta ATPases and nucleotide-binding proteins of the HUP class. This points to an extensive radiation of HUP domains before the last universal common ancestor (LUCA), during which the multiple class I aminoacyl-tRNA synthetases emerged only at a late stage. Thus, substantial evolutionary diversification of protein domains occurred well before the modern version of the protein-dependent translation machinery was established, i.e., still in the RNA world.

Adenosine Triphosphatases↗
PubMed2003

Identification of proteins highly expressed in the hyphae of Candida albicans by two-dimensional electrophoresis.

The increase in Candida albicans infections is caused by the increase in therapies resulting in immunocompromised patients. One factor required for C. albicans pathogenicity is the morphological transition from yeast to hypha. The protein profiles of whole extracts from yeasts and hyphae were examined using two-dimensional electrophoresis to identify the proteins related to the morphological transition. Over 900 protein spots were visualized by silver staining and 11 spots were increased more than three-fold reproducibly during hyphal differentiation. Six of the 11 spots were identified by peptide mass fingerprints, of which three represented PRA1, two PHR1 and the last TSA1. Vertical streak patterns of Pra1p and Phr1p indicated that post-translational modifications seem to be caused by variable glycosylation. Comparative proteome analysis between the wild-type and the deletion mutants, CAMB43 (deltapra1) and CAS10 (deltaphr1), further confirmed the identity of PRA1 and PHR1. Interestingly, Pra1p was downregulated in phr1-deleted mutants. Only PHR1 transcription was increased, indicating that PRA1 and TSA1 are controlled at the post-translational level.

Apoenzymes↗
PubMed1995

A slot blot method for detection of ultraviolet photoproducts in DNA.

A simple new method was established to detect the major uv photoproducts in DNA. A slot blot method, involving the use of T4 DNA polymerase-associated (3'--> 5') exonuclease digestion of uv-irradiated DNA, was used to detect (primarily) cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) dimers. Hydrolysis of DNA by this enzyme is quantitatively blocked by both of these photoproducts. To detect (primarily) (6-4) dimers, Escherichia coli DNA photolyase was utilized to specifically reverse cyclobutane pyrimidine dimers.

DNA↗
PubMed1998

Evasion of UVC-induced apoptosis by photorepair of cyclobutane pyrimidine dimers.

Cyclobutyl pyrimidine dimer (CPD) photolyase is known to reverse pyrimidine dimers specifically under illumination with visible light. OCP13, a Medaka cell line showing a high level expression of the gene for CPD photolyase, completely reversed pyrimidine dimers induced by 20 J/m2 UVC by 1 h of photorepair. When OCP13 cells were irradiated with 20 J/m2 UVC, morphological changes such as shrinkage of cells, distorted nuclear shape, and decrease in the number of nucleoli appeared 2 to 4 h after UVC irradiation. Thereafter, the irradiated cells began to detach from the substratum, and DNA ladders were observed in the DNA extracted from detached cells. Thus, these changes in cells after UVC exposure were used to characterize the progression of UV-induced apoptosis in OCP13 cells. Although formation of DNA ladders and cell detachment were blocked by cycloheximide treatment prior to UVC exposure, the morphological changes were not. With photorepair treatment, even after the morphological changes appeared cells were still able to restore their normal morphological features and remained attached. On the other hand, the cell-cycle progression in UVC-irradiated cells was arrested even after photorepair of pyrimidine dimers. Thus, photorepair can rescue cells from UV-induced apoptosis, although DNA damage other than that of pyrimidine dimers, as well as additional non-DNA damage, possibly remained, and DNA replication was left inhibited. Among the various kinds of damage induced by UVC irradiation, the presence of pyrimidine dimers is proposed to be the major trigger for UVC-induced apoptosis.

Animals↗
PubMed1999

Characterization of a Neurospora crassa photolyase-deficient mutant generated by repeat induced point mutation of the phr gene.

We produced a photolyase-deficient mutant by repeat induced point mutation using the Neurospora crassa photolyase gene cloned previously. This mutation identified a new gene, phr, which was mapped on the right arm of linkage group I by both RFLP mapping and conventional mapping. To investigate the relationship between photoreactivation and dark repair processes, especially excision repair, double mutants of phr with representative repair-defective mutants of different types were constructed and tested for UV sensitivity and photoreactivation. The results show that the phr mutation has no influence on dark repair. Tests with CPD and TC(6-4) photoproduct-specific antibodies demonstrated that the phr mutant is defective in CPD photolyase and confirmed that there is no TC(6-4) photolyase activity in N. crassa. Furthermore, N. crassa photolyase is not a blue light receptor in the signal transduction that induces carotenoid biosynthesis.

Blotting, Southern↗
PubMed1996

Cloning, tissue expression, and mapping of a human photolyase homolog with similarity to plant blue-light receptors.

Enzymatic photoreactivation is a DNA repair mechanism that removes UV-induced pyrimidine dimer lesions by action of a single enzyme, photolyase, and visible light. Its presence has been demonstrated in a wide variety of organisms, ranging from simple prokaryotes to higher eukaryotes. We have isolated a human gene encoding a 66-kDa protein that shows clear overall homology to known bacterial photolyase genes. The human gene product is more similar to plant blue-light receptors within class I photolyases than to higher eukaryote class II photolyases. Northern blot analysis showed two transcripts with constitutive expression in all tissues examined and an elevated expression in testis. In situ hybridization with a cDNA-derived probe localized this human gene to chromosome 12q23-q24.1. Southern analysis of the cloned human gene suggests a wide distribution of the gene family in various species.

Amino Acid Sequence↗