Mechanisms of DNA excision repair.
Explore the source record for details and available documents.
Aging↗
Biomedical subjects
A Sancar
Publications and source records attributed to A Sancar.
Substrate spectrum of human excinuclease: repair of abasic sites, methylated bases, mismatches, and bulky adducts.
Nucleotide-excision repair is the repair system for removing bulky lesions from DNA. Humans deficient in this repair pathway suffer from xeroderma pigmentosum (XP), a disease characterized by photodermatoses, including skin cancers. At the cellular level, XP patients fail to remove cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts induced by UV light, as well as other bulky DNA lesions caused by various genotoxic agents. XP cells are not particularly sensitive to ionizing radiation or to alkylating agents that cause mostly nonbulky DNA lesions. Therefore, it has generally been assumed that the human nucleotide-excision repair enzyme (excinuclease) is specific for bulky adducts. To determine the substrate range of human excinuclease we used the highly sensitive excision assay and tested bulky adducts, synthetic apurinic/apyrimidinic sites, N6-methyladenine, O6-methylguanine, and mismatches as potential substrates. We found that all of these "lesions" were removed by human excinuclease, although with vastly different efficiencies.
Animals↗
Human and E.coli excinucleases are affected differently by the sequence context of acetylaminofluorene-guanine adduct.
Synthetic DNA substrates containing an acetylaminofluorene (AAF) adduct at each of the three guanine in the G1G2CG3CC sequence were constructed and tested as substrates for reconstituted E.coli (A)BC excinuclease and human excinuclease in HeLa cell-free extract (CFE). The (A)BC excinulcease repaired the three substrates with relative efficiencies of G1:G2:G3 of 100:18:66 in agreement with an earlier report [Seeberg, E., and Fuchs, R.P.P. (1990) Proc. Natl Acad. Sci. USA 87, 191-194]. The same lesions were repaired by the human excinuclease with the strikingly different efficiencies of G1:G2:G3 as 38:100:68. These results reveal that the human excinuclease is affected by the sequence context of the lesion in a different manner than its prokaryotic counterpart.
Base Sequence↗
HMG-domain proteins specifically inhibit the repair of the major DNA adduct of the anticancer drug cisplatin by human excision nuclease.
The most frequent DNA adduct made by the anticancer drug cisplatin, the 1,2-intrastrand d(GpG) cross-link, as well as the minor 1,3-intrastrand d(GpTpG) adduct, were both repaired by an in vitro human excision repair system. Fragments of 27-29 nt containing the platinum damage were excised. The high mobility group (HMG)-domain proteins HMG1 and human mitochondrial transcription factor specifically inhibited repair of the 1,2-intrastrand cross-link by the human excision nuclease. These results suggest that the types and levels of HMG-domain proteins in a given tumor may influence the responsiveness of that cancer to cisplatin chemotherapy and they provide a rational basis for the synthesis of new platinum anticancer drug candidates.
Base Sequence↗
Flow linear dichroism and electron microscopic analysis of protein-DNA complexes of a mutant UvrB protein that binds to but cannot kink DNA.
(A)BC excinuclease of Escherichia coli is the enzymatic activity resulting from sequential and partially overlapping actions of UvrA, UvrB, and UvrC protein. UvrA is a molecular matchmaker which promotes the formation of a stable UvrB-damaged DNA complex in which the DNA is kinked by about 130 degrees. The UvrB-DNA complex is then recognized by UvrC and two incisions are made in the DNA by the joint actions of UvrC and UvrB. A mutant of UvrB (D478A) can be loaded onto the DNA but it does not interact with UvrC to cause a nick 3' to the lesion. Based on the lack of a DNase-I-hypersensitive site in the footprint of the mutant, it was proposed that the lack of incision was due to the inability of the mutant UvrB to kink the DNA. In the current study we have investigated the interaction of the mutant UvrB with DNA using two biophysical methods, flow linear dichroism and electron microscopy. Both methods reveal that the mutant UvrB is unable to bend DNA.
Adenosine Triphosphatases↗
Characterization of a medium wavelength type DNA photolyase: purification and properties of photolyase from Bacillus firmus.
The gene for the apoenzyme of Bacillus firmus photolyase was cloned and sequenced. The enzyme was overproduced in Escherichia coli, purified, and characterized. It has the unique property of having the maximum activity over a wavelength range where all other known photolyases exhibit modest activity. The enzyme contains reduced FAD and methenyltetrahydrofolate and has an absorption and action spectrum peak at 410 nm, and it repairs DNA with a quantum yield of phi approximately 0.75.
Amino Acid Sequence↗
Determination of minimum substrate size for human excinuclease.
Human cells remove bulky adducts from DNA by excising single-stranded fragments 27-29 nucleotides in length by an enzyme system consisting of at least 14 polypeptides. All of the previous work on characterizing the excision reaction was conducted with plasmids 3 or 8 kilobases in length. To determine if the size and tertiary structure of DNA play a role in the excision reaction and to find out if large DNA fragments are necessary to contact all of the subunits of the excinuclease, we performed experiments with circular DNA and with linear DNA fragments of various sizes. We found that the human excinuclease is capable of removing DNA adducts from linear and covalently closed circular DNAs with about the same efficiency. Furthermore, we found that the excinuclease can remove a thymine dimer or a psoralen-thymine monoadduct from linear fragments provided that the distance between the lesion and the 5'-terminus of the damaged strand is > or = 60 nucleotides and the distance between the lesion and the 3'-terminus is > or = 44 nucleotides. Thus, the minimum size substrate for human excinuclease is approximately 100 base pairs in length.
Base Sequence↗
Molecular cloning of the human nucleotide-excision-repair gene ERCC4.
ERCC4 was previously identified in somatic cell hybrids as a human gene that corrects the nucleotide-excision-repair deficiency in mutant hamster cells. The cloning strategy for ERCC4 involved transfection of the repair-deficient hamster cell line UV41 with a human sCos-1 cosmid library derived from chromosome 16. Enhanced UV resistance was seen with one cosmid-library transformant and two secondary transformants of UV41. Cosmid clones carrying a functional ERCC4 gene were isolated from a library of a secondary transformant by selecting in Escherichia coli for expression of a linked neomycin-resistance gene that was present in the sCos-1 vector. The cosmids mapped to 16p13.13-p13.2, the location assigned to ERCC4 by using somatic cell hybrids. Upon transfection into UV41, six cosmid clones gave partial correction ranging from 30% to 64%, although all appeared to contain the complete gene. The capacity for in vitro excision of thymine dimers from a plasmid by transformant cell extracts correlated qualitatively with enhanced UV resistance.
Animals↗
Formation of a ternary complex by human XPA, ERCC1, and ERCC4(XPF) excision repair proteins.
The xeroderma pigmentosum complementation group A (XP-A) protein, XPA, has recently been expressed in Escherichia coli in a soluble and fully functional form. An affinity column was prepared by linking the XPA protein to a solid support. When HeLa cell-free extract capable of excision repair was applied to the column, > 99.9% of the proteins were in the flow-through. However, the flow-through fraction lacked excision activity. The activity was restored by adding the high salt (1 M KCl) eluate of the column to the flow-through fraction. The XPA protein-bound fraction was tested for specific proteins by an in vitro complementation assay with a panel of cell-free extracts from DNA repair-deficient human and rodent cell lines. The XPA-bound fraction complemented cell-free extracts of excision repair cross-complementing 1 (ERCC-1), ERCC-4 (XP-F), and XP-A mutants. We conclude that the XPA damage recognition protein makes a ternary complex with the ERCC1/ERCC4(XPF) heterodimer with a potential nuclease function.
Animals↗
Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II.
The RNA polymerase II general transcription factor TFIIH is composed of several polypeptides. The observation that the largest subunit of TFIIH is the excision-repair protein XPB/ERCC3 (ref. 1), a helicase implicated in the human DNA-repair disorders xeroderma pigmentosum (XP) and Cockayne's syndrome, suggests a functional link between transcription and DNA repair. To understand the connection between these two cellular processes, we have extensively purified and functionally analysed TFIIH. We find that TFIIH has a dual role, being required for basal transcription of class II genes and for participation in DNA-excision repair. TFIIH is shown to complement three different cell extracts deficient in excision repair: XPB/ERCC3, XPC and XPD/ERCC2. The complementation of XPB and XPD is a consequence of ERCC3 and ERCC2 being integral subunits of TFIIH, whereas complementation of XPC is due to an association of this polypeptide with TFIIH. We found that the general transcription factor IIE negatively modulates the helicase activity of TFIIH through a direct interaction between TFIIE and the ERCC3 subunit of TFIIH.
DNA Helicases↗
Where transcription meets repair.
Explore the source record for details and available documents.
Animals↗
Characterization of (6-4) photoproduct DNA photolyase.
Pyrimidine (6-4) pyrimidone photoproduct is the second most abundant UV photoproduct in DNA. Recently, it was reported that Drosophila melanogaster cell-free extracts restored the biological activity of (6-4) photo-product-containing DNA in a light-dependent reaction (Todo, T., Takemori, H., Ryo, H., Ihara, M., Matsunaga, T., Nikaido, O., Sato, K., and Nomura, T. (1993) Nature 361, 372-374) concomitant with the loss of (6-4) photoproduct antigenic sites and (6-4) photoproduct-caused alkali-labile sites. In the present study we show that the (6-4) photoproduct but not its Dewar isomer is the substrate for the enzyme, that the enzyme has an action spectrum peak at 400 nm, and that the efficiency of repair per incident photon is very low compared with cyclobutane pyrimidine dimer photolyases. Furthermore, we provide evidence that the (6-4) photoproduct photolyase converts the photoproduct to unmodified bases probably through an oxetane intermediate.
Animals↗
Structure and function of DNA photolyase.
Cyclobutane pyrimidine dimers (Pyr < > Pyr) are the major DNA photoproducts induced by the UV component of solar radiation. Photoreactivating enzyme (DNA photolyase) repairs DNA by utilizing the energy of visible light to break the cyclobutane ring of the dimer. Photolyases are monomeric proteins of 50-60 kDa with stoichiometric amounts of two noncovalent chromophore/cofactors. One of these cofactors is FADH-, and the second chromophore is either methenyltetrahydrofolate (MTHF) or 8-hydroxy-5-deazariboflavin (8-HDF). The enzyme binds the DNA substrate in a light-independent reaction, the second chromophore of the bound enzyme absorbs a visible photon and, by dipole-dipole interaction, transfers energy to FADH- which, in turn, transfers an electron to Pyr < > Pyr in DNA; the Pyr < > Pyr- splits and back electron transfer restores the dipyrimidine and the functional form of flavin ready for a new cycle of catalysis.
Apoenzymes↗
Mechanisms of transcription-repair coupling and mutation frequency decline.
Mutation frequency decline is the rapid and irreversible decline in the suppressor mutation frequency of Escherichia coli cells if the cells are kept in nongrowth media immediately following the mutagenic treatment. The gene mfd, which is necessary for mutation frequency decline, encodes a protein of 130 kDa which couples transcription to excision repair by binding to RNA polymerase and to UvrA, which is the damage recognition subunit of the excision repair enzyme. Although current evidence suggests that transcription-repair coupling is the cause of the preferential repair of the transcribed strand of mRNA encoding genes as well as of suppressor tRNA genes, the decline occurs under stringent response conditions in which the tRNA genes are not efficiently transcribed. Thus, the mechanism of strand-specific repair is well understood, but some questions remain regarding the precise mechanism of mutation frequency decline.
Animals↗
Reconstitution of mammalian excision repair activity with mutant cell-free extracts and XPAC and ERCC1 proteins expressed in Escherichia coli.
Nucleotide excision repair in humans involves the coordinated actions of 8-10 proteins. To understand the roles of each of these proteins in excision it is necessary to develop an in vitro excision repair system reconstituted entirely from purified proteins. Towards this goal we have expressed in E. coli two of the 8 genes known to be essential for the excision reaction. XPAC and ERCC1 were expressed as fusion proteins with the Escherichia coli maltose binding protein (MBP) and purified to > 80% homogeneity by affinity chromatography. The purified proteins either as fusions or after cleavage from the MBP were able to complement the CFE of cells with mutations in the corresponding genes in an excision assay with thymine dimer containing substrate.
ATP-Binding Cassette Transporters↗
Comparative analysis of binding of human damaged DNA-binding protein (XPE) and Escherichia coli damage recognition protein (UvrA) to the major ultraviolet photoproducts: T[c,s]T, T[t,s]T, T[6-4]T, and T[Dewar]T.
Human cells contain a protein that binds to UV-irradiated DNA with high affinity. This protein, the damaged DNA-binding protein (DDB), is absent from some xeroderma pigmentosum complementation group E cell strains; therefore, it has been suggested that it may be the damage recognition subunit of a human excision nuclease complex. However, the identity of the UV photoproduct bound by DDB and the role of this protein in nucleotide excision repair have been controversial. In this study, we used several synthetic DNA substrates, each of which contains one of the major UV photoproducts, and DDB purified to apparent homogeneity to quantify the specific binding of DDB to various photoproducts. For comparison, the binding of the same photoproducts by the Escherichia coli damage recognition protein UvrA, which is known to be a subunit of the E. coli excision nuclease, was also measured. UvrA and DDB each bound with high affinity to T[t,s]T, T[6-4]T, and T[Dewar]T, but only marginally discriminated between an undamaged oligomer and an oligomer with a T[c,s]T. In contrast to these similarities with regard to the binding to UV photoproducts, UvrA bound to another excision repair substrate, the psoralen-thymine monoadduct, with high specificity, whereas DDB was unable to distinguish between psoralen-adducted DNA and undamaged DNA. We conclude that DDB may play a special role in the repair of UV damage, but it cannot be the sole damage recognition subunit of human excision nuclease.
Adenosine Triphosphatases↗
Time-resolved EPR studies with DNA photolyase: excited-state FADH0 abstracts an electron from Trp-306 to generate FADH-, the catalytically active form of the cofactor.
Photolyase repairs UV-induced cyclobutane-pyrimidine dimers in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains 5,10-methenyltetrahydrofolate, which functions as the light-harvesting chromophore, and fully reduced flavin adenine dinucleotide (FAD), which functions as the redox catalyst. During enzyme preparation, the flavin is oxidized to FADH0, which is catalytically inert. Illumination of the enzyme with 300- to 600-nm light converts the flavin to the fully reduced form in a reaction that involves photooxidation of an amino acid in the apoenzyme. The results of earlier optical studies had indicated that the redox-active amino acid in this photoactivation process was tryptophan. We have now used time-resolved electron paramagnetic resonance (EPR) spectroscopy to investigate the photoactivation reaction. Excitation of the flavin-radical-containing inactive enzyme produces a spin-polarized radical that we identify by 2H and 15N labeling as originating from a tryptophan residue, confirming the inferences from the optical work. These results and Trp-->Phe replacement by site-directed mutagenesis reveal that flavin radical photoreduction is achieved by electron abstraction from Trp-306 by the excited-state FADH0. Analysis of the hyperfine couplings and spin density distribution deduced from the isotopic-labeling results shows that the product of the light-driven redox chemistry is the Trp-306 cation radical. The results strongly suggest that the active form of photolyase contains FADH- and not FADH2.
Amino Acid Sequence↗
DNA photolyase repairs the trans-syn cyclobutane thymine dimer.
DNA photolyases catalyze the splitting of the cyclobutane ring joining the two dihydropyrimidines of a pyrimidine dimer by a photoinduced electron-transfer reaction. Previous studies concluded that photolyase repairs only the cis-syn form of the eight stereoisomers of the cyclobutane pyrimidine dimer (Pyr[ ]Pyr). In this study we found that Escherichia coli photolyase binds to the trans-syn-I isomer of T[ ]T with about 10(4)-fold lower affinity than the cis-syn isomer but it repairs it relatively efficiently.
Base Sequence↗