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A Sancar

Publications and source records attributed to A Sancar.

At least 163 records · Page 9Linked to original sources
PubMed1990

Mechanisms of caffeine inhibition of DNA repair in E. coli.

Caffeine inhibits excision repair and photoreactivation in E. coli in vivo. We used purified E. coli enzymes and DNase I footprinting to study the mechanism of inhibition in vitro. Photolyase binds to pyrimidine dimers in DNA in a radiation-independent process. Upon irradiation of this enzyme-substrate complex with photoreactivating light, pyrimidine dimers are reverted to their constituent pyrimidine monomers. Using an oligonucleotide containing a thymine dimer at a unique site, we found that caffeine associates with the substrate and inhibits photoreactivation by blocking the binding of photolyase to the dimer. ABC excinuclease catalyses early events of excision repair; recognition of covalently modified DNA and incision of the phosphodiester backbone on both sides of the modification. The UvrA subunit is involved in the damage recognition process, which we studied using an oligonucleotide containing a unique psoralen adduct. UvrA binds to the adduct and protects 33 base pairs surrounding the adduct from DNase I digestion. In the presence of caffeine, the DNaseI footprint of UvrA covers the entire oligonucleotide; thus, caffeine promotes the binding of UvrA to undamaged DNA. UvrA subunits "trapped" by caffeine would be unable to catalyze repair. The intercalators ethidium bromide and chloroquine also promoted UvrA binding to DNA, so it may be caffeine's ability to intercalate into DNA that results in the trapping of UvrA. Thus, as a consequence of its interaction with DNA, caffeine inhibits these repair systems in E. coli by two entirely different mechanisms, by promoting the nonspecific binding of the nucleotide excision repair enzyme and by interfering with specific binding of the photoreactivating enzyme.

Bacterial Proteins↗
PubMed1989

Preferential DNA repair of (6-4) photoproducts in the dihydrofolate reductase gene of Chinese hamster ovary cells.

We have developed a method to quantify (6-4) photoproducts in genes and other specific sequences within the genome. This approach utilizes the following two enzymes from Escherichia coli: ABC excinuclease, a versatile DNA repair enzyme which recognizes many types of lesions in DNA, and DNA photolyase, which reverts pyrimidine dimers. DNA is isolated from UV irradiated Chinese hamster ovary cells and digested with a restriction enzyme. Pyrimidine dimers, the major photoproduct produced at biological UV fluences, are then completely repaired by treatment with DNA photolyase. The photoreactivated DNA is treated with ABC excinuclease, electrophoresed in an alkaline agarose gel, transferred to a support membrane and probed for specific genomic sequences. Net incisions produced by ABC excinuclease following photoreactivation are largely due to the presence of (6-4) photoproducts. These adducts are quantitated by measuring the reduction of intensity of the full length fragments on the autoradiogram. Using this approach we have shown that (6-4) photoproducts are produced at equal frequency in the dihydrofolate reductase coding sequence and in its 3'-flanking, noncoding sequences and that the formation of (6-4) photoproducts is linear in both sequences up to a UV dose of 60 J/m2. The repair of (6-4) photoproducts in these DNA sequences was measured after a dose of 40 J/m2 over 4-, 8-, and 24-h time periods. The (6-4) photoproducts are repaired more efficiently than pyrimidine dimers in both sequences and there is preferential repair of (6-4) photoproducts in the dihydrofolate reductase gene compared with the downstream, noncoding sequences.

Animals↗
PubMed1989

A new mechanism for repairing oxidative damage to DNA: (A)BC excinuclease removes AP sites and thymine glycols from DNA.

Escherichia coli (A)BC excinuclease is the major enzyme responsible for removing bulky adducts, such as pyrimidine dimers and 6-4 photoproducts, from DNA. Mutants deficient in this enzyme are extremely sensitive to UV and UV-mimetic agents, but not to oxidizing agents, or ionizing radiation which damages DNA in part by generating active oxygen species. DNA glycosylases and AP1 endonucleases play major roles in repairing oxidative DNA damage, and thus it has been assumed that nucleotide excision repair has no role in cellular defense against damage by ionizing radiation and oxidative damage. In this study we show that the E. coli nucleotide excision repair enzyme (A)BC excinuclease removes from DNA the two major products of oxidative damage, thymine glycol and the baseless sugar (AP site). We conclude that nucleotide excision repair is an important cellular defense mechanism against oxidizing agents.

DNA Damage↗
PubMed1989

Evidence from extended X-ray absorption fine structure and site-specific mutagenesis for zinc fingers in UvrA protein of Escherichia coli.

The UvrA protein is the damage recognition subunit of the Escherichia coli repair enzyme ABC excision nuclease. Sequence analysis of this 940-amino acid protein revealed two regions of sequence homology to the zinc finger motif found in many DNA binding proteins. Physical and chemical analyses indicate about 2 zinc atoms/molecule. We have used extended x-ray absorption fine structure analysis to demonstrate that each of these zinc atoms is coordinated with 4 cysteine residues at a distance of 2.32 +/- 0.2 A. Substitution of one of the cysteines by a histidine, a serine, or an alanine in one of the potential finger sites resulted in a respective decrease in complementing activity. We thus conclude that the two zinc fingers identified by sequence analysis do indeed have zinc finger structure in UvrA protein.

Bacterial Proteins↗
PubMed1989

Purification and properties of Methanobacterium thermoautotrophicum DNA photolyase.

We have purified DNA photolyase from the autotrophic anaerobic archaebacterium Methanobacterium thermoautotrophicum to near homogeneity by a two-column affinity chromatography. The purified enzyme has an Mr = 60,000 and shows near UV absorption peak at 440 nm and a fluorescence emission maximum at 462 nm indicating that it contains 8-hydroxy-5-deazaflavin (coenzyme F420) as an intrinsic chromophore. The photolyase binds with high specificity to thymine dimer in DNA with an equilibrium binding constant, KA = 1.4 x 10(9) M-1, and a dissociation rate constant, koff = 1.4 x 10(-4) s-1 (t1/2 = 43 min). Despite 6-fold higher affinity compared to the folate-containing Escherichia coli photolyase the two enzymes apparently contact the same phosphates around the thymine dimer: the phosphate immediately 5' and the three phosphates immediately 3' to the dimer on the damaged strand and the phosphate across from the dimer in the minor groove on the complementary strand. The absolute action spectrum of the Methanobacterium photolyase in the 400-500-nm region closely matches the absorption of the enzyme-bound F420. The quantum yield (phi) over this region is constant and is approximately 0.2. The value is measurably smaller than the quantum yields reported for other DNA photolyases.

Amino Acid Sequence↗
PubMed1989

Human nucleotide excision repair in vitro: repair of pyrimidine dimers, psoralen and cisplatin adducts by HeLa cell-free extract.

We searched for nucleotide excision repair in human cell-free extracts using two assays: damage-specific incision of DNA (the nicking assay) and damage-stimulated DNA synthesis (the repair synthesis assay). HeLa cell-free extract prepared by the method of Manley et al. (1980) has a weak nicking activity on UV irradiated DNA and the nicking is only slightly reduced when pyrimidine dimers are eliminated from the substrate by DNA photolyase. In contrast to the nicking assay, the extract gives a strong signal with UV irradiated substrate in the repair synthesis assay. The repair synthesis activity is ATP dependent and is reduced by about 50% by prior treatment of the substrate with DNA photolyase indicating that this fraction of repair synthesis is due to removal of pyrimidine dimers by nucleotide excision. Psoralen and cisplatin adducts which are known to be removed by nucleotide excision repair also elicited repair synthesis activity 5-10 fold above the background synthesis. When M13RF DNA containing a uniquely placed psoralen adduct was used in the reaction, complete repair was achieved in a fraction of molecules as evidenced by the restoration of psoralen inactivated KpnI restriction site. This activity is absent in xeroderma pigmentosum group A cells. We conclude that our cell-free extract contains the human nucleotide excision repair enzyme activity.

Cell-Free System↗
PubMed1989

Role of enzyme-bound 5,10-methenyltetrahydropteroylpolyglutamate in catalysis by Escherichia coli DNA photolyase.

DNA photolyase catalyzes the photoreversal of pyrimidine dimers. The enzymes from Escherichia coli and yeast contain a flavin chromophore and a folate cofactor, 5,10-methenyltetrahydropteroylpolyglutamate. E. coli DNA photolyase contains about 0.3 mol of folate/mol flavin, whereas the yeast photolyase contains the full complement of folate. E. coli DNA photolyase is reconstituted to a full complement of the folate by addition of 5,10-methenyltetrahydrofolate to cell lysates or purified enzyme samples. The reconstituted enzyme displays a higher photolytic cross section under limiting light. Treatment of photolyase with sodium borohydride or repeated camera flashing results in the disappearance of the absorption band at 384 nm and is correlated with the formation of modified products from the enzyme-bound 5,10-methenyltetrahydrofolate. Photolyase modified in this manner has a decreased photolytic cross section under limiting light. Borohydride reduction results in the formation of 5,10-methylenetetrahydrofolate and 5-methyltetrahydrofolate, both of which are released from the enzyme. Repeated camera flashing results in photodecomposition of the enzyme-bound 5,10-methenyltetrahydrofolate and release of the decomposition products. Finally, it is observed that photolyase binds 10-formyltetrahydrofolate and appears to cyclize it to form the 5,10-methenyltetrahydrofolate chromophore.

Borohydrides↗
PubMed1989

Repair of O6-methylguanine by ABC excinuclease of Escherichia coli in vitro.

O6-Methylguanine, the major mutagenic product of methylnitroso compounds, was previously thought to be repaired exclusively by alkyltransferases I and II. Using synthetic substrates that contain O6-methyl-guanine at defined positions, we demonstrate that the nucleotide excision repair enzyme of Escherichia coli, ABC excinuclease, also repairs DNA containing this adduct. We show that the ABC excinuclease binds specifically to the modified DNA and produces incisions at the eight phosphodiester bond 5' and at the fifth or sixth phosphodiester bond 3' to the modified guanine.

Bacterial Proteins↗
PubMed1989

The LexA protein does not bind specifically to the two SOS box-like sequences immediately 5' to the phr gene.

There are two SOS box-like sequences located at the regions -161 to -142 and -69 to -50 with regard to the initiation codon of phr. Ihara et al. (1987) constructed a phr'-'lacZ fusion plasmid in which these sequences and the amino terminal end of the phr gene was fused to lacZ and therefore lacZ was under the regulatory control of phr promoter-operator. The authors found that in cells carrying this plasmid beta-galactosidase was inducible by UV and UV-mimetic agents and concluded that phr was controlled by the LexA repressor. We wished to confirm these results by theoretical analysis of the SOS-like sequences by the method developed by Berg (1987) as well as by measuring the binding of LexA protein to the putative SOS boxes by a novel gel retardation assay. Both theoretical analysis and experimental results indicate that the putative SOS boxes immediately 5' to phr have no specific affinity for LexA protein.

Bacterial Proteins↗
PubMed1989

The (A)BC excinuclease of Escherichia coli has only the UvrB and UvrC subunits in the incision complex.

The uvrA, uvrB, and uvrC genes control excision repair in Escherichia coli. Cells with mutations in any of these three genes cannot repair DNA by nucleotide excision. When the purified gene products--the UvrA, UvrB, and UvrC proteins--are mixed together, an excision nuclease is formed that incises on both sides of the damaged nucleotide in an ATP-dependent reaction; it has been presumed that the excision nuclease was an ABC complex containing all three Uvr proteins. To determine the stoichiometry of the subunits in the enzyme, we conducted hydrodynamic studies with mixtures of the subunits with or without DNA substrate. We found that without DNA the UvrA subunit is a dimer and that when UvrB protein is also present, a (UvrA)2(UvrB)1 complex forms. Without DNA no detectable interaction of either the UvrA or UvrB subunits or the (UvrA)2(UvrB)1 complex with the UvrC subunit occurs. Unexpectedly, with UV-irradiated DNA, the UvrA/UvrB ratio in isolated DNA-protein complexes is variable, and the ratio becomes infinitesimally low as the UvrA concentration in the reaction mixture decreases. Under conditions of saturating UvrB protein approximately one UvrB molecule binds to DNA per damaged site in a reaction that requires catalytic amounts of UvrA subunit. Addition of UvrC protein to purified UvrB-DNA complexes results in rapid incision of the DNA, presumably catalyzed by an excision nuclease containing only UvrB and UvrC subunits.

Bacterial Proteins↗
PubMed1988

Analysis of sequential steps of nucleotide excision repair in Escherichia coli using synthetic substrates containing single psoralen adducts.

Escherichia coli ABC excinuclease initiates the removal of dodecanucleotides from damaged DNA in an ATP-dependent reaction. Using a synthetic DNA fragment containing a psoralen adduct at a defined position we have investigated the interaction of the components of the enzyme with substrate by DNase I footprinting. We find that the UvrA subunit binds to DNA specifically in the absence of cofactors and that the binding affinity is stimulated about 4-fold by ATP and only marginally inhibited by ADP. The UvrA.DNA complexes formed in the absence of co-factors or in the presence of either ATP or ADP are remarkably similar. In contrast, adenosine 5'-O-(thiotriphosphate) increases nonspecific binding and completely abolishes the UvrA footprint. The UvrB subunit can associate with the UvrA subunit on DNA in the absence of ATP, but this ternary UvrA.UvrB.DNA complex is qualitatively different from that formed in the presence of ATP. The UvrC subunit elicits no additional change in the UvrA-UvrB footprint. Helicase II (UvrD protein) does not alter the UvrA-UvrB footprint but does appear to interact at the 5'-incision site of the postincision complex. DNA polymerase I fills in the excision gap in the presence or absence of helicase II and apparently releases the ABC excinuclease from the repaired DNA. Nearly 90% of the repair patches are 12 nucleotides long, and this length is not affected by helicase II. We see no evidence by DNase I footprinting for the formation of a multiprotein complex encompassing the UvrA, -B, -C, and -D proteins and DNA polymerase I.

Adenosine Diphosphate↗
PubMed1988

Use of psoralen-modified oligonucleotides to trap three-stranded RecA-DNA complexes and repair of these cross-linked complexes by ABC excinuclease.

A series of site-specifically cross-linked, three-stranded complexes has been prepared using the RecA protein, form I (covalently closed and negatively supercoiled) pUC19 plasmid, and eight psoralen-monoadducted oligonucleotides between 30 and 107 residues in length. Complexes formed much less efficiently if linearized pUC19 was used as the duplex substrate. Quantitative analysis indicates that although RecA is able to utilize a 30-mer in its homologous pairing reaction, incorporation at 50% efficiency requires a single-stranded substrate at least 50 residues long. These three-stranded complexes have been used to study the action mechanism for cross-link repair by ABC excinuclease, and the results are consistent with the recent model of Van Houten, B., Gamper, H., Holbrook, S. R., Hearst, J. E., and Sancar, A. (1986b) Proc. Natl. Acad. Sci. U. S. A. 83, 8077-8081.

Bacterial Proteins↗
PubMed1988

ABC excinuclease incises both 5' and 3' to the CC-1065-DNA adduct and its incision activity is stimulated by DNA helicase II and DNA polymerase I.

CC-1065 is a large molecule that binds covalently to adenine residues of DNA in a sequence-specific manner and lies in the minor groove about four bases to the 5' side of the adducted residue. Using a reconstituted Escherichia coli nucleotide excision repair system, we have obtained data showing that the ABC excinuclease makes incisions both 5' and 3' to the CC-1065 adduct and that the incision activity is stimulated by the addition of helicase II and DNA polymerase I (and dNTPs). Our results with the CC-1065 adduct are consistent with the reported in vitro processing of other adducts (e.g., cisplatin, UV photoproducts) but do not agree with a recent study that reported anomalous processing of the CC-1065 adduct by ABC excinuclease and helicase II. Our results also imply that, in binding to damaged DNA, ABC excinuclease does not make important contacts in the minor groove four bases to the 5' side of the damaged residue.

Adenosine Triphosphatases↗
PubMed1988

General method for quantifying base adducts in specific mammalian genes.

A general method has been developed to measure the formation and removal of DNA adducts in defined sequences of mammalian genomes. Adducted genomic DNA is digested with an appropriate restriction enzyme, treated with Escherichia coli UvrABC excision nuclease (ABC excinuclease), subjected to alkaline gel electrophoresis, and probed for specific sequences by Southern hybridization. The ABC excinuclease incises DNA containing bulky adducts and thus reduces the intensity of the full-length fragments in Southern hybridization in proportion to the number of adducts present in the probed sequence. This method is similar to that developed by Bohr et al. [Bohr, V. A., Smith, C. A., Okumoto, D. S. & Hanawalt, P. C. (1985) Cell 40, 359-369] for quantifying pyrimidine dimers by using T4 endonuclease V. Because of the wide substrate range of ABC exinuclease, however, our method can be used to quantify a large variety of DNA adducts in specific genomic sequences.

4-Nitroquinoline-1-oxide↗
PubMed1988

Identification of the second chromophore of Escherichia coli and yeast DNA photolyases as 5,10-methenyltetrahydrofolate.

Denaturation of DNA photolyase (deoxyribodipyrimidine photolyase, EC 4.1.99.3) from Escherichia coli with guanidine hydrochloride or acidification to pH 2 released, in addition to FAD, a chromophore with the spectral and chromatographic properties of a reduced pterin. Treatment of the enzyme with iodine prior to acidification converted the chromophore to a stable, oxidized derivative, which was resolved by HPLC into four species with identical spectral properties. The same species, in the same distribution, were obtained from the yeast enzyme. The material isolated from the iodine-oxidized enzyme was shown to be a pterin by conversion to pterin-6-carboxylic acid with alkaline permanganate and was found to release glutamate upon acid hydrolysis. The presence of 10-formylfolate in the isolated, oxidized chromophore was demonstrated by absorption and fluorescence spectroscopy and by deformylation and conversion to folic acid. Analysis of the distribution of polyglutamates revealed that the four species identified by HPLC corresponded to the tri-, tetra-, penta-, and hexaglutamate derivatives of 10-formylfolate. The results were consistent with gamma linkages in the triglutamate derivative with additional glutamates linked via the alpha-carboxyl group of the preceding residue. Treatment with rat plasma hydrolase produced the monoglutamate derivative of 10-formylfolate. The native, enzyme-bound form of the folate cofactor was identified as 5,10-methenyltetrahydrofolylpolyglutamate by effecting release and isolation at low pH to protect the 5,10-methenyl bridge and preserve the reduced pyrazine ring structure.

Bacterial Proteins↗
PubMed1988

Thymine dimers bend DNA.

A 32-base-pair DNA fragment containing a thymine photodimer was constructed and ligated head-to-tail to obtain multimers of this sequence in which thymine dimers were in phase with the helix screw axis (approximately equal to 3 turns apart). The ligation products were analyzed by one- and two-dimensional gel electrophoresis and quantitative electron microscopy. These analyses show that the thymine photodimer introduces a bend of approximately equal to 30 degrees in DNA, which causes anomalously slow migration of DNA fragments in polyacrylamide gels and facilitates the formation of small covalent circles. Repair of thymine dimers by DNA photolyase abolishes the anomalous migration.

DNA↗