Repair of N-methyl-N''-nitro-N-nitrosoguanidine-induced DNA damage by ABC excinuclease.

Escherichia coli has several overlapping DNA repair pathways which act in concert to eliminate the DNA damage caused by a diverse array of physical and chemical agents. The ABC excinuclease which is encoded by the uvrA, uvrB, and uvrC genes mediates both the incision and excision steps of nucleotide excision repair. Traditionally, this repair pathway has been assumed to be active against DNA adducts that cause major helical distortions. To determine the level of helical deformity required for recognition and repair by ABC excinuclease, we have evaluated the substrate specificity of this enzyme by using DNA damaged by N-methyl-N'-nitro-N-nitrosoguanidine. ABC excinuclease incised methylated DNA in vitro in a dose-dependent manner in a reaction that was ATP dependent and specific for the fully reconstituted enzyme. In vivo studies with various alkylation repair-deficient mutants indicated that the excinuclease participated in the repair of DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine.     

Action mechanism of Escherichia coli DNA photolyase. II. Role of the chromophores in catalysis

DNA photolyase repairs pyrimidine dimers in DNA in a reaction that requires visible light. Photolyase from Escherichia coli is normally isolated as a blue protein and contains 2 chromophores: a blue FAD radical plus a second chromophore that exhibits an absorption maximum at 360 nm when free in solution. Oxidation of the FAD radical is accompanied by a reversible loss of activity which is proportional to the fraction of the enzyme flavin converted to FADox. Quantitative reduction of the radical to fully reduced FAD causes a 3-fold increase in activity. The results show that a reduced flavin is required for activity and suggest that flavin may act as an electron donor in catalysis. Comparison of the absorption spectrum calculated for the protein-bound second chromophore (lambda max = 390 nm) with fluorescence data and with the relative action spectrum for dimer repair indicates that the second chromophore is the fluorophore in photolyase and that it does act as a sensitizer in catalysis. On the other hand, enzyme preparations containing diminished amounts of the second chromophore do not exhibit correspondingly lower activity. This suggests that reduced flavin may also act as a sensitizer in catalysis. The blue color of the enzyme is lost upon reduction of the FAD radical. The fully reduced E. coli enzyme exhibits absorption and fluorescence properties very similar to yeast photolyase. This indicates that the two enzymes probably contain similar chromophores but are isolated in different forms with respect to the redox state of the flavin.     

Identification and amplification of the E. coli phr gene product.

We have constructed a series of multicopy plasmids that complement mutations in the phr gene of Escherichia coli. By subcloning into a tac plasmid vector we obtained a phr plasmid that upon induction overproduces two proteins of Mr's 49,000 and 20,000. Tn1000 insertions into the phr gene caused the disappearance of the 49,000 dalton protein, thus demonstrating this protein to be the phr gene product, DNA photolyase. The photolyase encoded by the phr gene makes up about 15% of total cellular proteins after induction of cells carrying a tac-phr plasmid. This protein binds specifically to UV (254 nm) irradiated DNA and upon exposure to near UV (300-500 nm) illumination repairs the UV damage and dissociates from DNA.     

Gene-specific formation and repair of cisplatin intrastrand adducts and interstrand cross-links in Chinese hamster ovary cells

We have used three methods to study the formation and repair of intrastrand adducts and interstrand cross-links in the DNA of Chinese hamster ovary cells induced by the anticancer drug cis-diamminedichloroplatinum II (cisplatin). Using atomic absorption spectroscopy, we found that 21% of the total genomic cisplatin adducts were removed at 8 h and 42% at 24 h. We used ABC excinuclease digestion, coupled with out previously reported methodology to quantify DNA in specific genomic regions. These adducts were removed faster in the transcribed dihydrofolate reductase and c-myc genes compared to a noncoding fragment, a region containing the little or nontranscribed c-fos oncogene, and to the overall genome. Interstrand cross-links in specific sequences were quantified by Southern hybridization of denatured-renatured DNA separated on a neutral gel. We found that cross-links were removed more efficiently from the gene regions than intrastrand adducts and, at high levels of cross-linking, removal was similar from transcribed and from nontranscribed regions.     

Post-incision steps of nucleotide excision repair in Escherichia coli. Disassembly of the UvrBC-DNA complex by helicase II and DNA polymerase I.

UvrA, UvrB, and UvrC initiate nucleotide excision repair by incising a damaged DNA strand on each side of the damaged nucleotide. This incision reaction is substoichiometric with regard to UvrB and UvrC, suggesting that both proteins remain bound following incision and do not "turn over." The addition of only helicase II to such reaction mixtures turns over UvrC; UvrB turnover requires the addition of helicase II, DNA polymerase I, and deoxynucleoside triphosphates. Column chromatography and psoralen photocross-linking experiments show that following incision, the damaged oligomer remains associated with the undamaged strand, UvrB, and UvrC in a post-incision complex. Helicase II releases the damaged oligomer and UvrC from this complex, making repair synthesis possible; DNase I footprinting experiments show that UvrB remains bound to the resulting gapped DNA until displaced by DNA polymerase I. The specific binding of UvrB to a psoralen adduct in DNA inhibits psoralen-mediated DNA-DNA cross-linking, yet promotes the formation of UrvB-psoralen-DNA cross-links. The discovery of psoralen-UvrB photocross-linking offers the potential of active-site labeling.     

Isolation and characterization of functional domains of UvrA.

The sequence of Escherichia coli UvrA protein suggests that it may fold into two functional domains each possessing DNA binding and ATPase activities. We have taken two approaches to physically isolate polypeptides corresponding to the two putative domains. First, a 180 base pair DNA segment encoding multiple collagenase recognition sequences was inserted into UvrA's putative interdomain hinge region. This UvrA derivative was purified and digested with collagenase, and the resulting 70-kDa N-terminal and 35-kDa C-terminal fragments were purified. Both fragments possessed nonspecific DNA binding activity, but only the N-terminal domain retained its nucleotide binding capacity as evidence by measurements of ATP hydrolysis and by ATP photo-cross-linking. Together, the two fragments failed to substitute for UvrA in reconstituting (A)BC excinuclease and, therefore, were presumed to be unable to load UvrB onto damaged DNA. Second, the DNA segments encoding the two domains were fused to the beta-galactosidase gene. The UvrA N-terminal domain-beta-galactosidase fusion protein was overproduced and purified. This fusion protein had ATPase activity, thus confirming that the amino-terminal domain does possess an intrinsic ATPase activity independent of any interaction with the carboxy terminus. Our results show that UvrA has two functional domains and that the specificity for binding to damaged DNA is provided by the proper three-dimensional orientation of one zinc finger motif relative to the other and is not an intrinsic property of an individual zinc finger domain.     

Site-specific mutagenesis of conserved residues within Walker A and B sequences of Escherichia coli UvrA protein.

UvrA is the ATPase subunit of the DNA repair enzyme (A)BC excinuclease. The amino acid sequence of this protein has revealed, in addition to two zinc fingers, three pairs of nucleotide binding motifs each consisting of a Walker A and B sequence. We have conducted site-specific mutagenesis, ATPase kinetic analyses, and nucleotide binding equilibrium measurements to correlate these sequence motifs with activity. Replacement of the invariant Lys by Ala in the putative A sequences indicated that K37 and K646 but not K353 are involved in ATP hydrolysis. In contrast, substitution of the invariant Asp by Asn in the B sequences at positions D238, D513, or D857 had little effect on the in vivo activity of the protein. Nucleotide binding studies revealed a stoichiometry of 0.5 ADP/UvrA monomer while kinetic measurements on wild-type and mutant proteins showed that the active form of UvrA is a dimer with 2 catalytic sites which interact in a positive cooperative manner in the presence of ADP; mutagenesis of K37 but not of K646 attenuated this cooperativity. Loss of ATPase activity was about 75% in the K37A, 86% in the K646A mutant, and 95% in the K37A-K646A double mutant. These amino acid substitutions had only a marginal effect on the specific binding of UvrA to damaged DNA but drastically reduced its ability to deliver UvrB to the damage site. We find that the deficient UvrB loading activity of these mutant UvrA proteins results from their inability to associate with UvrB in the form of (UvrA)2(UvrB)1 complexes. We conclude that UvrA forms a dimer with two ATPase domains involving K37 and K646 and that the work performed by ATP hydrolysis is the delivery of UvrB to the damage site on DNA.     

High-performance liquid chromatographic separation of platinum complexes containing the cis-1,2-diaminocyclohexane carrier ligand

Platinum drugs with the 1,2-diaminocyclohexane (dach) carrier ligand have shown great promise in cancer chemotherapy, but little is known about their metabolism in the body. Since it is possible to radiolabel the dach ligand, it should be possible to quantitate the biotransformation products of these drugs, provided a method were available to separate the biotransformation products. In this paper we describe a two-column high-performance liquid chromatography system which can be used to separate many likely dach-platinum biotransformation products from the parent compounds, and allow their identification. An initial separation on a reverse-phase Partisil ODS-3 column allowed resolution of the uncharged species. The peak fractions from this column were concentrated 10-fold and reinjected onto a cation exchange Partisil 10 SCX column to allow resolution of the positively charged species. This system allowed resolution of two prototype dach-platinum drugs, (cis-1,2-diaminocyclohexane)dichloroplatinum(II) and (cis-1,2-diaminocyclohexane)malonatoplatinum(II), the aquated species likely to form from these drugs, and the complexes formed when these compounds react with glutathione, metallothionein, and amino acids. By using cation exchange chromatography at pH 2.3 as well as pH 4 and by using 14C-labeled amino acids to determine stoichiometry, it was also possible to determine the most likely structures for some of the amino acid complexes. Most importantly, this system allowed clear separation of many of the likely biotransformation products tested from the biologically important aquated species. This system should prove useful for separating and identifying the biotransformation products of dach-platinum drugs in blood and urine, in tissue culture media, and inside the cell.     

(A)BC excinuclease: the Escherichia coli nucleotide excision repair enzyme

Nucleotide excision repair is the major pathway for removing damage from DNA. (A)BC excinuclease is the nuclease activity which initiates nucleotide excision repair in Escherichia coli. In this review, we focus on current understanding of the structure-function of the enzyme and the reaction mechanism of the repair pathway. In addition, recent biochemical studies on preferential repair of actively transcribed genes in E. coli are summarized.