Eker, A.P.M.

A baculovirus photolyase with DNA repair activity and circadian clock regulatory function (Article)

2012-02-01T00:00:00Z

Cryptochromes and photolyases belong to the same family of flavoproteins but, despite being structurally conserved, display distinct functions. Photolyases use visible light to repair ultraviolet-induced DNA damage. Cryptochromes, however, function as blue-light receptors, circadian photoreceptors, or repressors of the CLOCK/BMAL1 heterodimer, the transcription activator controlling the molecular circadian clock. Here, we present evidence that the functional divergence between cryptochromes and photolyases is not so univocal. Chrysodeixis chalcites nucleopolyhedrovirus possesses 2 photolyase-like genes: phr1 and phr2. We show that PHR1 and PHR2 are able to bind the CLOCK protein. Only for PHR2, however, the physical interaction with CLOCK represses CLOCK/BMAL1-driven transcription. This result shows that binding of photolyase per se is not sufficient to inhibit the CLOCK/BMAL1 heterodimer. PHR2, furthermore, affects the oscillation of immortalized mouse embryonic fibroblasts, suggesting that PHR2 can regulate the molecular circadian clock. These findings are relevant for further understanding the evolution of cryptochromes and photolyases as well as behavioral changes induced in insects by baculoviruses.

The potorous cpd photolyase rescues a cryptochrome-deficient mammalian circadian clock (Article)

2011-08-19T00:00:00Z

Despite the sequence and structural conservation between cryptochromes and photolyases, members of the cryptochrome/photolyase (flavo)protein family, their functions are divergent. Whereas photolyases are DNA repair enzymes that use visible light to lesion-specifically remove UV-induced DNA damage, cryptochromes act as photoreceptors and circadian clock proteins. To address the functional diversity of cryptochromes and photolyases, we investigated the effect of ectopically expressed Arabidopsis thaliana (6-4)PP photolyase and Potorous tridactylus CPD-photolyase (close and distant relatives of mammalian cryptochromes, respectively), on the performance of the mammalian cryptochromes in the mammalian circadian clock. Using photolyase transgenic mice, we show that Potorous CPD-photolyase affects the clock by shortening the period of behavioral rhythms. Furthermore, constitutively expressed CPD-photolyase is shown to reduce the amplitude of circadian oscillations in cultured cells and to inhibit CLOCK/BMAL1 driven transcription by interacting with CLOCK. Importantly, we show that Potorous CPD-photolyase can restore the molecular oscillator in the liver of (clock-deficient) Cry1/Cry2 double knockout mice. These data demonstrate that a photolyase can act as a true cryptochrome. These findings shed new light on the importance of the core structure of mammalian cryptochromes in relation to its function in the circadian clock and contribute to our further understanding of the evolution of the cryptochrome/photolyase protein family.

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV (Article)

2011-06-07T00:00:00Z

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH-). The CPD repair mechanism involves electron transfer from photoexcited FADH-to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH-. The two electron transfer processes occur on time scales of 10-10and 10-9s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T○-) and electron return from T○-to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between <1 ps and <100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.

DNA photolyases of Chrysodeixis chalcites nucleopolyhedrovirus are targeted to the nucleus and interact with chromosomes and mitotic spindle structures (Article)

2010-04-01T00:00:00Z

Cyclobutane pyrimidine dimer (CPD) photolyases convert UV-induced CPDs in DNA into monomers using visible light as the energy source. Two phr genes encoding class II CPD photolyases PHR1 and PHR2 have been identified in Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV). Transient expression assays in insect cells showed that PHR1-EGFP fusion protein was localized in the nucleus. Early after transfection, PHR2-EGFP was distributed over the cytoplasm and nucleus but, over time, it became localized predominantly in the nucleus. Immunofluorescence analysis with anti-PHR2 antiserum showed that, early after transfection, non-fused PHR2 was already present mainly in the nucleus, suggesting that the fusion of PHR2 to EGFP hindered its nuclear import. Both PHR-EGFP fusion proteins strongly colocalized with chromosomes and spindle, aster and midbody structures during host-cell mitosis. When PHR2-EGFP- transfected cells were superinfected with Autographa californica multiplenucleocapsid NPV (AcMNPV), the protein colocalized with virogenic stroma, the replication factories of baculovirus DNA. The collective data support the supposition that the PHR2 protein plays a role in baculovirus DNA repair.

DNA repair by photolyase: A novel substrate with low background absorption around 265 nm for transient absorption studies in the UV (Article)

2010-01-19T00:00:00Z

CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidine dimer (CPD), by photoreversion of the damage reaction. An enzyme-bound reduced flavin (FADH-) cofactor functions as photosensitizer. Upon excitation, it transiently transfers an electron to the CPD, triggering scission of the interpyrimidine bonds. After repair completion, the electron returns to the flavin to restore its functional reduced form. A major difficulty for time-resolved spectroscopic monitoring of the enzymatic repair reaction is that absorption changes around 265 nm accompanying pyrimidine restoration are obscured by the strong background absorption of the nondimerized bases in DNA. Here we present a novel substrate for CPD photolyase that absorbs only weakly around 265 nm: a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3′ end, replaced by 5,6-dihydrothymine which virtually does not absorb around 265 nm. Repair of this substrate by photolyases from Anacystis nidulans and from Escherichia coli was compared with repair of two conventional substrates: a 10-mer of unmodified thymidines containing a central CPD and an acetone-sensitized thymidine 18-mer that contained in average six randomly distributed CPDs per strand. In all cases, the novel substrate was repaired with an efficiency very similar to that of the conventional substrates (quantum yields in the order of 0.5 upon excitation of FADH-). Flash-induced transient absorption changes at 267 nm could be recorded on a millisecond time scale with a single subsaturating flash and yielded very similar signals for all three substrates. Because of its low background absorption around 265 nm and the defined structure, the novel substrate is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase.

Very fast product release and catalytic turnover of DNA photolyase (Article)

2009-07-20T00:00:00Z

Direct DNA damage reversal: Elegant solutions for nasty problems (Article)

2009-03-01T00:00:00Z

The genomic integrity of all living organisms is constantly jeopardized by physical [e.g. ultraviolet (UV) light, ionizing radiation] and chemical (e.g. environmental pollutants, endogenously produced reactive metabolites) agents that damage the DNA. To overcome the deleterious effects of DNA lesions, nature evolved a number of complex multi-protein repair processes with broad, partially overlapping substrate specificity. In marked contrast, cells may use very simple repair systems, referred to as direct DNA damage reversal, that rely on a single protein, remove lesions in a basically error-free manner, show high substrate specificity, and do not involve incision of the sugar-phosphate backbone or base excision. This concise review deals with two types of direct DNA damage reversal: (i) the repair of alkylating damage by alkyltransferases and dioxygenases, and (ii) the repair of UV-induced damage by spore photoproduct lyases and photolyases. (Part of a Multi-author Review).

What makes the difference between a cryptochrome and DNA photolyase? A spectroelectrochemical comparison of the flavin redox transitions (Article)

2009-01-21T00:00:00Z

Cryptochromes and DNA photolyases are highly homologous flavoproteins that accomplish completely different tasks. While plant cryptochrome1 functions as blue light photoreceptor that triggers various morphogenic reactions, photolyases repair UV-induced DNA damages. Both enzymes share the photoactive cofactor, noncovalently bound FAD. For photolyase, the reaction mechanism involves electron transfer to the substrate from the excited-state of fully reduced flavin. For cryptochrome, photoexcitation of the oxidized flavin leads to formation of the semireduced radical FADḢ. Key parameters for the redox state of the flavin in the cell are the midpoint potentials E1 and E2 for the oxidized/semireduced and semireduced/fully reduced transitions, respectively. A link between cryptochrome function and its cofactor's redox states has been suggested early on, but no reliable determinations of midpoint potentials have been available. Here we report spectroelectrochemical titrations of cryptochrome1 from Arabidopsis thaliana and photolyases from both E. coli and Anacystis nidulans at pH 7.4. For the cryptochrome, we obtained E1 ≈ E2 ≈ -160 mV vs NHE, strongly deviating from the photolyases where FADḢ could not be oxidized up to 400 mV, and E2 ≈ -40 mV. Functional and evolutionary implications are discussed, highlighting the role of an asparagine-to-aspartate replacement close to N5 of the flavin.

Active DNA photolyase encoded by a baculovirus from the insect Chrysodeixis chalcites (Article)

2008-08-02T00:00:00Z

The genome of Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) contains two open reading frames, Cc-phr1 and Cc-phr2, which encode putative class II CPD-DNA photolyases. CPD-photolyases repair UV-induced pyrimidine cyclobutane dimers using visible light as an energy source. Expression of Cc-phr2 provided photolyase deficient Escherichia coli cells with photoreactivating activity indicating that Cc-phr2 encodes an active photolyase. In contrast, Cc-phr1 did not rescue the photolyase deficiency. Cc-phr2 was overexpressed in E. coli and the resulting photolyase was purified till apparent homogeneity. Spectral measurements indicated the presence of FAD, but a second chromophore appeared to be absent. Recombinant Cc-phr2 photolyase was found to bind specifically F0 (8-hydroxy-7,8-didemethyl-5-deazariboflavin), which is an antenna chromophore present in various photolyases.. After reconstitution, FAD and F0 were present in approximately equimolar amounts. In reconstituted photolyase the F0 chromophore is functionally active as judged from the increase in the in vitro repair activity. This study demonstrates for the first time that a functional photolyase is encoded by an insect virus, which may have implications for the design of a new generation of baculoviruses with improved performance in insect pest control.

50 years thymine dimer (Article)

2008-03-01T00:00:00Z

Fifty years ago thymine dimer was discovered in the Biochemical and Biophysical Laboratory of Delft Technological University, The Netherlands, by one of the authors of this review (Beukers) as the first environmentally induced DNA lesion. It is one of the photoproducts formed between adjacent pyrimidine bases in DNA by UV irradiation, currently known as cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts. Major lesions found in DNA after in vitro or in vivo UV irradiation are the cis-syn cyclobutane thymine dimer and the thymine-cytosine (6-4) photoproduct. Even after 50 years the study of photo-induced DNA lesions is still going on as is illustrated by the hundreds of papers published every year and the millions hits when browsing the internet for dimer-related information. Living organisms possess efficient and different mechanisms to repair detrimental lesions in their DNA. A unique mechanism to repair CPDs is reversion by either direct interaction with light of short wavelength or by enzymatic photoreactivation. Photophysical mechanisms that induce and reverse molecular bonds in biological macromolecules have been a main focus of research of the group in Delft in the middle of the last century. This review describes the break-through results of these studies which were the result of intense interactions between scientists in the fields of physics, organic chemistry and biochemistry. Philosophically, the "view" of the group in Delft was very appealing: since in nature photolesions are induced in DNA by the sun, how is it possible that repair of these lesions could be accomplished by the same energy source. Evolutionary, it is hardly possible to think of a more efficient repair mechanism.

Observation of an intermediate tryptophanyl radical in W306F mutant DNA photolyase from Escherichia coli supports electron hopping along the triple tryptophan chain (Article)

2007-09-04T00:00:00Z

DNA photolyases repair UV-induced cyclobutane pyrimidine dimers in DNA by photoinduced electron transfer. The redox-active cofactor is FAD in its doubly reduced state FADH-. Typically, during enzyme purification, the flavin is oxidized to its singly reduced semiquinone state FADH°. The catalytically potent state FADH-can be reestablished by so-called photoactivation. Upon photoexcitation, the FADH° is reduced by an intrinsic amino acid, the tryptophan W306 in Escherichia coli photolyase, which is 15 Å distant. Initially, it has been believed that the electron passes directly from W306 to excited FADH°, in line with a report that replacement of W306 with redox-inactive phenylalanine (W306F mutant) suppressed the electron transfer to the flavin [Li, Y. F., et al. (1991) Biochemistry 30, 6322-6329]. Later it was realized that two more tryptophans (W382 and W359) are located between the flavin and W306; they may mediate the electron transfer from W306 to the flavin either by the superexchange mechanism (where they would enhance the electronic coupling between the flavin and W306 without being oxidized at any time) or as real redox intermediates in a three-step electron hopping process (FADH°* ← W382 ← W359 ← W306). Here we reinvestigate the W306F mutant photolyase by transient absorption spectroscopy. We demonstrate that electron transfer does occur upon excitation of FADH° and leads to the formation of FADH-and a deprotonated tryptophanyl radical, most likely W359°. These photoproducts are formed in less than 10 ns and recombine to the dark state in ∼1 μs. These results support the electron hopping mechanism.

Electron hopping through the 15 Å triple tryptophan molecular wire in DNA photolyase occurs within 30 ps (Article)

2006-08-17T00:00:00Z

Photoreduction of the semi-reduced flavin adenine dinucleotide cofactor FADḢ in DNA photolyase from Escherichia coli into FADH - involves three tryptophan (W) residues that form a closely spaced electron-transfer chain FADḢ-W382-W359-W306. To investigate this process, we have constructed a mutant photolyase in which W359 is replaced by phenylalanine (F). Monitoring its photoproducts by femtosecond spectroscopy, the excited-state FADḢ* was found to decay in ∼30 ps, similar as in wild type (WT) photolyase. In contrast to WT, however, in W359F mutant photolyase the ground-state FADḢ fully recovered virtually concomitantly with the decay of its excited state and, despite the presence of the primary electron donor W382, no measurable flavin reduction was observed at any time. Thus, W359F photolyase appears to behave like many other flavoproteins, where flavin excited states are quenched by very short-lived oxidation of aromatic residues. Our analysis indicates that both charge recombination of the primary charge separation state FADH-W382 ̇+ and (in WT) electron transfer from W359 to W382 ̇+ occur with time constants <4 ps, considerably faster than the initial W382→FADḢ* electron-transfer step. Our results provide a first experimental indication that electron transfer between aromatic residues can take place on the time scale of ∼10-12 s.

Enhanced repair of cyclobutane pyrimidine dimers and improved UV resistance in photolyase transgenic mice (Article)

2002-01-01T00:00:00Z

During evolution, placental mammals appear to have lost cyclobutane pyrimidine dimer (CPD) photolyase, an enzyme that efficiently removes UV-induced CPDs from DNA in a light-dependent manner. As a consequence, they have to rely solely on the more complex, and for this lesion less efficient, nucleotide excision repair pathway. To assess the contribution of poor repair of CPDs to various biological effects of UV, we generated mice expressing a marsupial CPD photolyase transgene. Expression from the ubiquitous beta-actin promoter allowed rapid repair of CPDs in epidermis and dermis. UV-exposed cultured dermal fibroblasts from these mice displayed superior survival when treated with photoreactivating light. Moreover, photoreactivation of CPDs in intact skin dramatically reduced acute UV effects like erythema (sunburn), hyperplasia and apoptosis. Mice expressing the photolyase from keratin 14 promoter photo reactivate CPDs in basal and early differentiating keratinocytes only. Strikingly, in these animals, the anti-apoptotic effect appears to extend to other skin compartments, suggesting the presence of intercellular apoptotic signals. Thus, providing mice with CPD photolyase significantly improves repair and uncovers the biological effects of CPD lesions.

Novel functional interactions between nucleotide excision DNA repair proteins influencing the enzymatic activities of TFIIH, XPG, and ERCC1-XPF (Article)

2001-01-09T00:00:00Z

The multisubunit basal transcription factor IIH (TFIIH) has a dual involvement in nucleotide excision repair (NER) of a variety of DNA lesions, including UV-induced photoproducts, and RNA polymerase II transcription. In both processes, TFIIH is implicated with local DNA unwinding, which is attributed to its helicase subunits XPB and XPD. To further define the role of TFIIH in NER, functional interactions between TFIIH and other DNA repair proteins were analyzed. We show that the TFIIH-associated ATPase activity is stimulated by both XPA and the XPC-HR23B complex. However, while XPA promotes the ATPase activity specifically in the presence of damaged DNA, stimulation by XPC-HR23B is lesion independent. Furthermore, we reveal that TFIIH inhibits the structure-specific endonuclease activities of both XPG and ERCC1-XPF, responsible for the 3' and 5' incision in NER, respectively. The inhibition occurs in the absence of ATP and is reversed upon addition of ATP. These results point toward additional roles for TFIIH and ATP during NER distinct from a requirement for DNA unwinding in the regulation of the endonuclease activities of XPG and ERCC1-XPF.

A human homolog of the yeast nucleotide excision repair gene MMS19 interacts with transcription repair factor TFIIH through the XPB and XPD helicases. (Article)

2000-01-01T00:00:00Z

Nucleotide excision repair (NER) removes UV-induced photoproducts and numerous other DNA lesions in a highly conserved 'cut-and-paste' reaction that involves approximately 25 core components. In addition, several other proteins have been identified which are dispensable for NER in vitro but have an undefined role in vivo and may act at the interface of NER and other cellular processes. An intriguing example is the Saccharomyces cerevisiae Mms19 protein that has an unknown dual function in NER and RNA polymerase II transcription. Here we report the cloning and characterization of a human homolog, designated hMMS19, that encodes a 1030 amino acid protein with 26% identity and 51% similarity to S.cerevisiae Mms19p and with a strikingly similar size. The expression profile and nuclear location are consistent with a repair function. Co-immunoprecipitation experiments revealed that hMMS19 directly interacts with the XPB and XPD subunits of NER-transcription factor TFIIH. These findings extend the conservation of the NER apparatus and the link between NER and basal transcription and suggest that hMMS19 exerts its function in repair and transcription by interacting with the XPB and XPD helicases.

Cloning of a human homolog of the yeast nucleotide excision repair gene MMS19 and interaction with transcription repair factor TFIIH via the XPB and XPD helicases (Article)

2000-01-01T00:00:00Z

MammalianCry1 and Cry2 are essential for maintenance of circadian rhythms (Letter To Editor)

1999-04-15T00:00:00Z

Many biochemical, physiological and behavioural processes show circadian rhythms which are generated by an internal time-keeping mechanism referred to as the biological clock. According to rapidly developing models, the core oscillator driving this clock is composed of an autoregulatory transcription-(post) translation-based feedback loop involving a set of 'dock' genes. Molecular clocks do not oscillate with an exact 24-hour rhythmicity but are entrained to solar day/night rhythms by light. The mammalian proteins Cryl and Cry2, which are members of the family of plant blue-light receptors (cryptochromes) and photolyases, have been proposed as candidate light receptors for photoentrainment of the biological clock. Here we show that mice lacking the Cryl or Cry2 protein display accelerated and delayed free-running periodicity of locomotor activity, respectively. Strikingly, in the absence of both proteins, an instantaneous and complete loss of free-running rhythmicity is observed. This suggests that, in addition to a possible photoreceptor and antagonistic clock-adjusting function, both proteins are essential for the maintenance of circadian rhythmicity.

Xeroderma pigmentosum group C complex is the initiator of global genome repair. (Miscellaneous)

1998-08-01T00:00:00Z

The XPC-HR23B complex is specifically involved in global genome but not transcription-coupled nucleotide excision repair (NER). Its function is unknown. Using a novel DNA damage recognition-competition assay, we identified XPC-HR23B as the earliest damage detector to initiate NER: it acts before the known damage-binding protein XPA. Coimmunoprecipitation and DNase I footprinting show that XPC-HR23B binds to a variety of NER lesions. These results resolve the function of XPC-HR23B, define the first NER stages, and suggest a two-step mechanism of damage recognition involving damage detection by XPC-HR23B followed by damage verification by XPA. This provides a plausible explanation for the extreme damage specificity exhibited by global genome repair. In analogy, in the transcription-coupled NER subpathway, RNA polymerase II may take the role of XPC. After this subpathway-specific initial lesion detection, XPA may function as a common damage verifier and adaptor to the core of the NER apparatus.

Two human homologs of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity. (Article)

1997-01-01T00:00:00Z

XPC-hHR23B protein complex is specifically involved in nucleotide excision repair (NER) of DNA lesions on transcriptionally inactive sequences as well as the nontranscribed strand of active genes. Here we demonstrate that not only highly purified recombinant hHR23B (rhHR23B) but also a second human homolog of the Saccharomyces cerevisiae Rad23 repair protein, hHR23A, stimulates the in vitro repair activity of recombinant human XPC (rhXPC), revealing functional redundancy between these human Rad23 homologs. Coprecipitation experiments with His-tagged rhHR23 as well as sedimentation velocity analysis showed that both rhHR23 proteins in vitro reconstitute a physical complex with rhXPC. Both complexes were more active than free rhXPC, indicating that complex assembly is required for the stimulation. rhHR23B was shown to stimulate an early stage of NER at or prior to incision. Furthermore, both rhHR23 proteins function in a defined NER system reconstituted with purified proteins, indicating direct involvement of hHR23 proteins in the DNA repair reaction via interaction with XPC.

XPC and human homologs of RAD23: intracellular localization and relationship to other nucleotide excision repair complexes. (Article)

1996-01-01T00:00:00Z

The xeroderma pigmentosum syndrome complementation group C (XP-C) is due to a defect in the global genome repair subpathway of nucleotide excision repair (NER). The XPC protein is complexed with HHR23B, one of the two human homologs of the yeast NER protein, RAD23 (Masutani at al. (1994) EMBO J. 8, 1831-1843). Using heparin chromatography, gel filtration and native gel electrophoresis we demonstrate that the majority of HHR23B is in a free, non-complexed form, and that a minor fraction is tightly associated with XPC. In contrast, we cannot detect any bound HHR23A. Thus the HHR23 proteins may have an additional function independent of XPC. The fractionation behaviour suggests that the non-bound forms of the HHR23 proteins are not necessary for the core of the NER reaction. Although both HHR23 proteins share a high level of overall homology, they migrate very differently on native gels, pointing to a difference in conformation. Gel filtration suggests the XPC-HHR23B heterodimer resides in a high MW complex. However, immunodepletion studies starting from repair-competent Manley extracts fall to reveal a stable association of a significant fraction of the HHR23 proteins or the XPC-HHR23B complex with the basal transcription/repair factor TFIIH, or with the ERCC1 repair complex. Consistent with a function in repair or DNA/chromatin metabolism, immunofluorescence studies show all XPC, HHR23B and (the free) HHR23A to reside in the nucleus.

Different removal of ultraviolet photoproducts in genetically related xeroderma pigmentosum and trichothiodystrophy diseases. (Article)

1995-01-01T00:00:00Z

To understand the heterogeneity in genetic predisposition to skin cancer in different nucleotide excision repair-deficient human syndromes, we studied repair of cyclobutane pyrimidine dimers (CPDs) and of pyrimidine(6-4)pyrimidone (6-4PP) photoproducts in cells from trichothiodystrophy (TTD) patients. TTD is not associated with increased incidence of skin cancer, although 50% of the patients are photosensitive and carry a defect in the nucleotide excision repair pathway, similar to Xeroderma pigmentosum patients. However, in striking contrast to TTD, Xeroderma pigmentosum is highly prone to cancer. To address this apparent paradox, two types of studies were conducted: (a) reactivation of UV-irradiated plasmids harboring actively transcribed reporter genes, with or without photolyase treatment before transfection of SV40-transformed fibroblasts; and (b) the kinetics of removal of UV-induced CPDs and 6-4PPs in genomic DNA by immunoblot analysis using lesion-specific mAbs in SV40-transformed and untransformed fibroblasts representative of all genetic TTD complementation groups. Results showed that all cell lines from photosensitive TTD patients efficiently express Cat or luciferase genes in transfected plasmids carrying non-CPD lesions, including 6-4PP, and display wild-type or near-wild-type (50-70% in 3 cell lines) 6-4PP repair in the overall genome after immunoblot analysis. However, CPD lesions (the repair of which is defective in the overall genome) also block the expression of the reporter gene in transfected plasmids. Two cell lines from nonphotosensitive TTD patients showed wild-type levels of repair for both photoproducts in overall genome. A model on the lesion-specific repair in the context of the molecular defect in TTD is proposed. The implication of the defective CPD repair and efficient 6-4PP repair subpathways in cancer prevention in TTD patients is discussed.

Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage binding protein. (Article)

1994-01-01T00:00:00Z

Cells from a subset of patients with the DNA-repair-defective disease xeroderma pigmentosum complementation group E (XP-E) are known to lack a DNA damage-binding (DDB) activity. Purified human DDB protein was injected into XP-E cells to test whether the DNA-repair defect in these cells is caused by a defect in DDB activity. Injected DDB protein stimulated DNA repair to normal levels in those strains that lack the DDB activity but did not stimulate repair in cells from other xeroderma pigmentosum groups or in XP-E cells that contain the activity. These results provide direct evidence that defective DDB activity causes the repair defect in a subset of XP-E patients, which in turn establishes a role for this activity in nucleotide-excision repair in vivo.

Xeroderma pigmentosum group A correcting protein from Calf Thymus. (Article)

1992-09-01T00:00:00Z

A proteinous factor was purified from calf thymus and HeLa cells, which specifically corrects the excision repair defect of xeroderma pigmentosum complementation group A (XP-A) cells. Recovery of UV-induced unscheduled DNA synthesis after microinjection of XP-A cells was used as a quantitative assay for the correcting activity of protein preparations. XP-A correcting protein appears to be very stable as it withstands heating to 100 degrees C and treatment with SDS or 6 M urea. A molecular weight of 40-45 kD was found both under native (gel filtration) and denaturing (SDS-PAGE) conditions. Calf XP-A protein binds to single-stranded DNA more strongly than to double-stranded DNA, but shows no clear preference for UV-irradiated DNA. Polyclonal antibodies raised against human recombinant XP-A protein, which strongly inhibit UV-induced unscheduled DNA synthesis of normal human cells, completely abolished XP-A correcting activity when mixed with calf thymus preparations. This indicates a close relationship between human gene product and the calf protein. In the final preparation two main protein bands were present. Only one band at approx. 41 kD showed both DNA binding activity in Southwestern blots and immune reaction with human XP-A antibody, suggesting that this is the active calf XP-A correcting factor.

Effects of microinjected photoreactivating enzyme on thymine dimer removal and DNA repair synthesis in normal human and xeroderma pigmentosum fibroblasts. (Article)

1990-01-01T00:00:00Z

UV-induced thymine dimers (10 J/m2 of UV-C) were assayed in normal human and xeroderma pigmentosum (XP) fibroblasts with a monoclonal antibody against these dimers and quantitative fluorescence microscopy. In repair-proficient cells dimer-specific immunofluorescence gradually decreased with time, reaching about 25% of the initial fluorescence after 27 h. Rapid disappearance of dimers was observed in cells which had been microinjected with yeast photoreactivating enzyme prior to UV irradiation. This photoreactivation (PHR) was light dependent and (virtually) complete within 15 min of PHR illumination. In general, PHR of dimers strongly reduces UV-induced unscheduled DNA synthesis (UDS). However, when PHR was applied immediately after UV irradiation, UDS remained unchanged initially; the decrease set in only after 30 min. When PHR was performed 2 h after UV exposure, UDS dropped without delay. An explanation for this difference is preferential removal of some type(s) of nondimer lesions, e.g., (6-4) photoproducts, which is responsible for the PHR-resistant UDS immediately following UV irradiation. After the rapid removal of these photoproducts, the bulk of UDS is due to dimer repair. From the rapid effect of dimer removal by PHR on UDS it can be deduced that the excision of dimers up to the repair synthesis step takes considerably less than 30 min. Also in XP fibroblasts of various complementation groups the effect of PHR was investigated. The immunochemical dimer assay showed rapid PHR-dependent removal comparable to that in normal cells. However, the decrease of (residual) UDS due to PHR was absent (in XP-D) or much delayed (in XP-A and -E) compared to normal cells. This supports the idea that in these XP cells preferential repair of nondimer lesions does occur, but at a much lower rate.

Use of in vivo and in vitro assays for the characterization of mammalian excision repair and isolation of repair proteins. (Article)

1990-01-01T00:00:00Z

Elucidation of the molecular mechanism of mammalian nucleotide excision repair requires the availability of purified proteins, DNA substrates with defined lesions and suitable repair assays. Repair assays introduced in recent years vary from testing individual steps and successions of steps in vitro to systems that closely reflect the entire process in vivo. In the first part of this review, an in vivo microinjection system is discussed. The second part of the article reviews an in vitro system for study of repair synthesis promoted by cell extracts. Both systems can be utilized as assays during the purification of protein factors that complement repair-defective xeroderma pigmentosum cells. The effect of purified repair proteins from other organisms on mammalian repair is also considered.

Unscheduled DNA synthesis in xeroderma pigmentosum cells after microinjection of yeast photoreactivating enzyme. (Article)

1986-03-01T00:00:00Z

Photoreactivating enzyme (PRE) from yeast causes a light-dependent reduction of UV-induced unscheduled DNA synthesis (UDS) when injected into the cytoplasm of repair-proficieint human fibroblasts (Zwetsloot et al., 1985). This result indicates that the exogenous PRE monomerizers UV-induced dimers in these cells competing with the endogenous excision repair. In this paper we present the results of the injection of yeast PRE on (residual) UDS in fibroblasts from different excision-deficient XP-strains representing complementation groups A, C, D, E, F, H and I (all displaying more than 10% of the UDS of wild-type cells) and in fibroblasts from two excision-proficient XP-variant strains. In fibroblasts belonging to complementation groups C, F and I and in fibroblasts from the XP-variant strains UDS was significantly reduced, indicating that pyrimidine dimers in these cells are accessible to and can be monomerized by the injected yeast PRE. The UDS reduction in the XP-variant strains is comparable with the effect in wild-type cells. In cells from complementation groups C, F and I the reduction is less than in wild-type and XP-variant cells. Fibroblasts belonging to groups A, D, E and H did not show any reduction in UDS level after PRE injection and illumination with photoreactivating light. These result give evidence that the genetic repair defect in some XP-strains is probably due to an altered accessibility of the UV-damaged sites.

Microinjected photoreactivating enzymes from Anacystis and Saccharomyces monomerize dimers in chromatin of human cells (Article)

1985-01-01T00:00:00Z

Photoreactivating enzymes (PRE) from the yeast Saccharomyces cerevisiae and the cyanobacterium Anacystis nidulans have been injected into the cytoplasm of repair-proficient human fibroblasts in culture. After administration of photoreactivation light, PRE-injected cells displayed a significantly lower level of UV-induced unscheduled DNA synthesis (UDS) than non-injected cells. This indicates that monomerization of the UV-induced pyrimidine dimers in the mammalian chromatin had occurred as a result of photoreactivation by the injected PRE at the expense of repair by the endogenous excision pathway. Purified PRE from yeast is able to reduce UDS to 20-25% of the UDS found in non-injected cells, whereas the in vitro more active PRE from A. nidulans gives a reduction to only 70%. This suggests that the eukaryotic enzyme is more efficient in the removal of pyrimidine dimers from mammalian chromatin than its equivalent purified from the prokaryote A. nidulans.