Differences and similarities in the repair of two benzo[a]pyrene diol epoxide isomers induced DNA adducts by uvrA, uvrB, and uvrC gene products.

We have determined the role of the uvrA, uvrB, and uvrC genes in Escherichia coli cells in repairing DNA damage induced by three benzo[a]pyrene diol epoxide isomers. Using the phi X174 RF DNA-E. coli transfection system, we have found that BPDE-I or BPDE-II modified phi X174 RF DNA has much lower transfectivity in uvrA, uvrB, and uvrC mutant cells compared to wild type cells. In contrast, BPDE-III modification of phi X174 RF DNA causes much less difference in transfectivity between wild type and uvr- mutant cells. Moreover, BPDE-I and -II-DNA adducts are much more genotoxic than are BPDE-III-DNA adducts. Using purified UVRA, UVRB, and UVRC proteins, we have found that these three gene products, working together, incise both BPDE-I- and BPDE-III-DNA adducts quantitatively and, more importantly, at the same rate. In general, UVRABC nuclease incises on both the 5' (six to seven nucleotides) and 3' (four nucleotides) sides of BPDE-DNA adducts with similar efficiency with few exceptions. Quantitation of the UVRABC incision bands indicates that both of these BPDE isomers have different sequence selectivities in DNA binding. These results suggest that although UVR proteins can efficiently repair both BPDE-I- and BPDE-III-DNA adducts, in vivo the uvr system is the major excision mechanism for repairing BPDE-I-DNA adducts but may play a lesser role in repairing BPDE-III-DNA adducts. It is possible the low lethality of BPDE-III-DNA adducts is due to less complete blockage of DNA replication.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recognition and repair of 2-aminofluorene- and 2-(acetylamino)fluorene-DNA adducts by UVRABC nuclease

Recognition of damage induced by N-hydroxy-2-aminofluorene (N-OH-AF) and N-acetoxy-2-(acetylamino)fluorene (NAAAF) in both phi X174 RFI supercoiled DNA and a linear DNA fragment by purified UVRA, UVRB, and UVRC proteins was investigated. We have previously demonstrated that N-OH-AF and NAAAF treatments produce N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene (dG-C8-AAF), respectively, in DNA. Using a piperidine cleavage method and DNA sequence analysis, we have found that all guanine residues can be modified by N-OH-AF and NAAAF. These two kinds of adducts have different impacts on the DNA helix structure; while dG-C8-AF maintains the anti configuration, dG-C8-AAF is in the syn form. phi X174 RF DNA-Escherichia coli transfection results indicate that while the uvrA, uvrB, and uvrC gene products are needed to repair dG-C8-AAF, the uvrC, but not the uvrA or uvrB gene products, is needed for repair of dG-C8-AF. However, we have found that in vitro the UVRA, UVRB, and UVRC proteins must work in concert to nick both dG-C8-AF and dG-C8-AAF. In general, the reactions of UVRABC nuclease toward dG-C8-AF are similar to those toward dG-C8-AAF; it incises seven to eight nucleotides from the 5' side and three to four nucleotides from the 3' side of the DNA adduct. Evidence is presented to suggest that hydrolysis on the 3' and 5' sides of the damaged base by UVRABC nuclease is not simultaneous and that at least occasionally hydrolysis occurs only on the 3' side or on the 5' side of the damage site. The possible mechanisms of UVRABC nuclease incision for AF-DNA are discussed.     

Recognition and repair of the CC-1065-(N3-adenine)-DNA adduct by the UVRABC nucleases

The recognition and repair of the helix-stabilizing and relatively nondistortive CC-1065-(N3-adenine)-DNA adduct by UVRABC nuclease has been investigated both in vivo with phi X174 RFI DNA by a transfection assay and in vitro by a site-directed adduct in a 117 base pair fragment from M13mp1. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis which binds within the minor groove of DNA through N3 of adenine. In contrast to the helix-destabilizing and distortive modifications of DNA caused by ultraviolet light or N-acetoxy-2-(acetylamino)fluorene, CC-1065 increases the melting point of DNA and decreases the S1 nuclease activity. Using a viral DNA-Escherichia coli transfection system, we have found that the uvrA, uvrB, and uvrC genes, which code for the major excision repair proteins for UV- and NAAAF-induced DNA damage, are also involved in the repair of CC-1065-DNA adducts. In contrast, the uvrD gene product, which has been found to be involved in the repair of UV damage, has no effect in repairing CC-1065-DNA adducts. Purified UVRA, UVRB, and UVRC proteins must work in concert to incise the drug-modified phi X174 RFI DNA. Using a site-directed and multiple CC-1065 modified (MspI-BstNI) 117 base pair fragment from M13mp1, we have found that UVRABC nuclease incises at the eighth phosphodiester bond on the 5' side of the CC-1065-DNA adduct on the drug-modified strand.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of the rates of reaction and function of UVRB in UVRABC- and UVRAB-mediated anthramycin-N2-guanine-DNA repair.

The repair of anthramycin-DNA adducts by the UVR proteins in Escherichia coli follows two pathways: the adducts may be incised by the combined actions of UVRA, UVRB, and UVRC, or alternatively, the anthramycin may be removed by UVRA and UVRB in the absence of UVRC and with no DNA strand incision. To assess the competition between these two competing pathways, the rate of UVRABC-mediated excision repair of anthramycin-N2-guanine DNA adducts and the rate of UVRAB-mediated removal of the adduct were measured with single end-labeled DNAs under identical reaction conditions. UVR protein concentrations of 15 nM UVRA, 100 nM UVRB, and 10 nM UVRC protein were chosen to mimic in vivo concentrations. With these UVR protein concentrations and anthramycin-DNA concentrations of 1-2 nM the incision reaction and the release reactions are described by first-order kinetics. The rate of the UVRABC reaction, measured as the increase in incised fragments, was six to seven times faster than the rate of the UVRAB reaction, measured as the decrease in incised fragments. The UVRABC incision rate on anthramycin-modified linear DNA was four to five times the incision rate measured on the same DNA irradiated with ultraviolet light. We also investigated the role of the ATPase function of UVRB in UVRAB-mediated anthramycin removal. We found that a UVRB analogue with alanine at arginine 51, which retains near wild type ATPase activity, supported removal of anthramycin in the presence of UVRA, whereas a UVRB analogue with alanine at lysine 45, which abolishes the ATPase activity, did not. UVRB*, a specific proteolytic cleavage product of UVRB which retains the ATPase activity, did support removal of anthramycin in the presence of UVRA.     

Recognition of the DNA helix stabilizing anthramycin-N2 guanine adduct by UVRABC nuclease

The binding of the anti-tumor antibiotic anthramycin to a defined linear DNA fragment was investigated using both exonuclease III and lambda exonuclease. We show that most of the guanine residues are reactive toward anthramycin; however, several guanine residues showed preferential reactivity for the drug. Using purified UVRA, UVRB and UVRC proteins we present evidence that these three proteins in concert are able to recognize and produce specific strand cleavage flanking anthramycin-DNA adducts. The cleavage of anthramycin adducts by UVRABC nuclease is specific and results in strand breaks at five or six bases 5' and three or four bases 3'-flanking an adduct. At some guanine residues single incisions were observed only on one side of the adduct. The 5' strand breaks observed often occurred as doublet bands on sequencing gels, indicating plasticity in the site of 5' cleavage whereas the 3' cleavage did not show this effect. When DNA fragments modified with elevated levels of anthramycin were used as substrates the activity of the UVRABC nuclease toward the anthramycin adducts decreased. Possible mechanisms for the recognition and specific cleavage of the helix-stabilizing anthramycin DNA adduct and other helix destabilizing lesions by the UVRABC nuclease are discussed.     

Reactions of the UVRABC excision nuclease with DNA damaged by diamminedichloroplatinum(II).

Mutants of Escherichia coli, which are blocked in excision repair (uvrA6, uvrB5, or uvrC34) are exceptionally sensitive to the antitumor drug cis-Pt(II)(NH3)2Cl2 (cis-DDP) but not the trans isomer. Plasmid DNA, damaged by either the cis or trans compound and treated with the UVRABC excision nuclease was cut as shown by conversion of supercoiled DNA to relaxed forms. All three protein products of the uvrA, uvrB, and uvrC genes were required for incision. End-labeled fragments damaged with cis-DDP and reacted with the UVRABC nuclease were cut at the 8th phosphodiester bond 5' and at the 4th phosphodiester bond 3' to adjacent GG's. DNA treated with trans-DDP was not cut appreciably at adjacent GG's by the repair enzyme as subsequent analysis of reaction products after enzyme digestion gave a pattern similar to those obtained with control untreated fragments. The results indicate that the UVRABC nuclease may promote cell survival by the removal of adjacent GG's which are crosslinked by cis-Pt(II)(NH3)2Cl2.     

Quantitation of a simian virus 40 nonhomologous recombination pathway.

We describe an infectious-center in situ plaque hybridization procedure which quantitates simian virus 40 (SV40) nonhomologous recombination in terms of the number of recombinant-producing cells in the DNA transfected cell population. Using this assay to measure the efficiency of recombination with SV40 DNA in permissive monkey BSC-1 cells, we found that: (i) over a range of DNA concentrations, polyomavirus DNA (which is partially homologous to SV40 DNA) cannot be distinguished from nonhomologous phi X174 RF1 DNA with respect to its ability to recombine with SV40 DNA; (ii) at defined DNA concentrations, polyomavirus and phi X174 RF1 DNA compete with each other for recombination with SV40 DNA; (iii) virtually all segments of the phi X174 genome recombine, apparently at random, with SV40 DNA; (iv) the frequency of recombinant-producing cells, among the successfully transfected (virion-producing) cells, depends upon the input SV40 DNA concentration in the transfection solution; and (v) replication-defective SV40 mutant DNAs compete with wild-type SV40 DNA for recombination with phi X174 RF1 DNA. From these observations, we conclude that the efficiency of recombination with SV40, in the system under study, is unaffected by nucleotide sequence homology and that a limiting stage in the recombination pathway occurs before SV40 DNA replication. Comparison of the dependency of recombination on initial SV40 DNA concentration with the dependency on initial phi X174 RF1 DNA concentration indicates that SV40 DNA sequences are a controlling factor in the nonhomologous recombination pathway.     

Immunochemical quantitation of thymine glycol in oxidized and X-irradiated DNA

The present study demonstrates the usefulness of immunochemical assays for quantitating modified bases in oxidized and X-irradiated DNA. Escherichia coli, phi X174 RF I, PM2, and M13 DNA containing thymine glycols introduced by OsO4 oxidation were used as antigens in a direct enzyme-linked immunosorbent assay (ELISA). The number of thymine glycols per DNA molecule was determined by reactivity with antithymine glycol antibody standardized either to the acetol fragment assay or to the number of Escherichia coli endonuclease III-sensitive sites. The number of thymine glycols was also determined in phi X174 RF I DNA X-irradiated in either phosphate or Tris buffer under air. Using a direct ELISA with phi X174 RF I DNA irradiated in a phosphate buffer solution, the anti-thymine glycol antibody detected damage at the level of 40 Gy. The immunochemical assay was sensitive, specific, quantitative, and independent of DNA structure.     

Single-strand breakage of DNA in UV-irradiated uvrA, uvrB, and uvrC mutants of Escherichia coli.

We transduced the uvrA6, uvrB5, uvrC34, and uvrC56 markers from the original mutagenized strains into an HF4714 background. Although in the original mutagenized strains uvrA6 cells are more UV sensitive than uvrB5 and uvrC34 cells, in the new background no significant difference in UV sensitivity is observed among uvrA6, uvrB5, and uvrC34 cells. No DNA single-strand breaks are detected in UV-irradiated uvrA6 or uvrB5 cells, whereas in contrast a significant number of single-strand breaks are detected in both UV-irradiated uvrC34 and uvrC56 cells. The number of single-strand breaks in these cells reaches a plateau at 20-J/m2 irradiation. Since these single-strand breaks can be detected by both alkaline sucrose and neutral formamide-sucrose gradient sedimentation, we concluded that the single-strand breaks observed in UV-irradiated uvrC cells are due to phosphodiester bond interruptions in DNA and are not due to apurinic/apyrimidinic sites.     

The A* protein of phi X174 is an inhibitor of DNA replication

Extracts prepared from phi X174 infected E. coli cells inhibited in vitro RF replication The inhibition was dependent upon the presence of A* protein in the reaction and served as an assay to highly purify the A* protein. Purified A* protein bound tightly to duplex DNA as well as single-stranded DNA. The binding of the A* protein to duplex DNA inhibited (I) its single-stranded DNA specific endonucleolytic activity; (II) in vitro synthesis of viral (+) single stranded DNA on an A-RFII DNA complex template; (III) ATP hydrolysis by rep protein and unwinding of the strands of RF DNA. We propose that this inhibitory activity is responsible in vivo for the shut off of E. coli chromosome replication during phi X174 infection, and has a role in the transition from semiconservative RF DNA replication to single-stranded DNA synthesis in the life cycle of phi X174.     

Occurrence and loss of alkali-labile sites in newly synthesized DNA of UV-irradiated Escherichia coli K12

In pulse-labelled DNA of ultraviolet-irradiated E. coli, alkali-labile sites were detected. They do not occur in undamaged cells. These sites are produced in wild-type cells as well as in uvrA, uvrB and recA derivatives. Restoration of the synthesis of DNA molecules free of alkali-labile sites requires recA products and involves also uvrA and uvrB products. The chemical nature of alkali-labile sites and their biological function are obscure. They might be stretches of RNA that traverse the lesions, blocking DNA replication and priming recA-dependent DNA replication.     

Properties and regulation of the UVRABC endonuclease

This report summarizes the cloning of the uvrA, uvrB and uvrC genes of E. coli, the identification and isolation of the gene products, the regulation of the genes, and reconstitution of active UVRABC endonuclease from the individually isolated components.     

A general approach for purifying proteins encoded by cloned genes without using a functional assay: isolation of the uvrA gene product from radiolabeled maxicells.

The uvrA protein (UVRA) of E. coli has been extensively purified from a strain in which UVRA is overproduced and specifically labeled with 35S-methionine. This approximately 100-fold overproduction relative to normal strains is a result of having the uvrA gene present on a multicopy plasmid in a spr recA cell that makes defective lexA protein, the normal repressor of the uvrA gene, while the specific labeling of UVRA is done with maxicells. This approach facilitates the preparation of the protein since enzyme assays do not have to be carried out during the intermediate stages of purification. The purified UVRA binds to DNA and has ATPase activity but does not have intrinsic endonuclease activity. When added to extracts of uvrA- cells, the purified UVRA does promote the specific cutting of UV-irradiated DNA. Since this approach for working out rapid purification procedures by specifically labeling the proteins encoded by cloned genes does not require the use of a functional assay, it is a general one that can be applied to a wide variety of other gene products in addition to UVRA.     

Equilibrium binding of carcinogens and antitumor antibiotics to DNA: site selectivity, cooperativity, allosterism.

The equilibrium binding of the carcinogens N-hydroxy-N-acetyl-2-amino-fluorene (HAAF) and 4-nitroquinoline-1-oxide (NQO) to phi X174RF DNA have been studied by phase partition techniques. Both molecules bind in a cooperative manner with only a few carcinogen molecules binding to each phi X174RF DNA molecule. The binding data for both HAAF and NQO fit a model in which two carcinogens cluster into a small number of sites--four sites for HAAF and twelve sites for NQO. Phase partition techniques were also used to study the binding of actinomycin D to both calf thymus DNA and poly (dG-dC) . poly (dG-dC) at much lower r values than had been previously reported. These data exhibit humped Scatchard plots which are indicative of cooperative binding; the overall shape of the Scatchard plots are consistent with a model for drug induced allosteric transitions in the DNA structure. The cooperativity in the actinomycin D binding to calf thymus DNA increases with decreasing sodium chloride concentration, suggesting a role for DNA flexibility in allosteric binding.     

DNA sequences which support activities of the bacteriophage phi X174 gene A protein

The DNA sequence of 30 nucleotides which surrounds the origin of viral strand DNA replication is highly conserved amongst the icosahedral single-stranded DNA bacteriophages. The A gene of these phages encodes a protein which is required for initiation and termination of viral strand DNA synthesis and acts as a nicking-closing activity specifically within this 30-nucleotide sequence. A system of purified Escherichia coli host proteins and phi X174 gene A protein has been developed which specifically replicates in vitro the viral strand of phi X174 from RF (replicative form) I template DNA and yields single-stranded circular DNA products (RF leads to SS(c) DNA replication system). Recombinant plasmids carrying inserts derived from phage phi X174 or G4 DNA which range in length from 49 to 1175 base pairs and contain the 30-nucleotide conserved sequence have been shown to support phi X A protein-dependent DNA synthesis in vitro in this replication system. We report here that insertion of the 30-nucleotide sequence alone into pBR322 allows the resulting recombinant plasmids to support phi X A protein-dependent in vitro DNA synthesis as efficiently as phi X174 template DNA in the RF leads to SS(c) replication system. The 30-nucleotide sequence functions as a fully wild type DNA replication origin as determined by the rate of DNA synthesis and the structure of resulting DNA products. Furthermore, the DNA sequence requirements for nicking of RF I DNA by the phi X A protein and for supporting replication origin function have been partially separated. Homology to positions 1, 29, and 30 of the 30-nucleotide conserved sequence are not required for cleavage of RF I DNA by the A protein; homology to position 1 but not 29 or 30 is required for efficient DNA replication.     

Process of attachment of phi X174 parental DNA to the host cell membrane

The phi X174-DNA membrane complex was isolated from Escherichia coli infected with phi X174 am3 by isopycnic sucrose gradient centrifugation followed by zone electrophoresis. The phi X174 DNA-membrane complex banded at two positions, intermediate density membrane fraction and cytoplasmic membrane fraction, having bouyant densities of 1.195 and 1.150 g/ml, respectively. Immediately after infection with phi X147, replicating DNA was pulse-labeled and then the incorporated label was chased. The radioactivity initially recovered in the intermediate density membrane fraction migrated to the cytoplasmic membrane fraction. The DNAs from both complexes sedimented mainly at the position of parental replicative form I (RFI). The phi X174 DNA-membrane complex contained a speficic membrane-bound protein having a molecular weigth of 80,000 which is accumulated in the host DNA-membrane complex. These results suggest that when phi X174 DNA penetrated into cells in the early phase of infection, single-stranded circular DNA was converted to parental RFI at a wall/membrane adhesion region and migrated to the cytoplasmic membrane fraction, where the parental RF could serve as a template in the replication of progeny RF.     

Structure of simian virus 40-phiX174 recombinant genomes isolated from single cells.

Three simian virus (SV40)-phi X174 recombinant genomes were isolated from single BSC-1 monkey cells cotransfected with SV40 and phi X174 RF1 DNAs. The individual cell progenies were amplified, cloned, and mapped by a combination of restriction endonuclease and heteroduplex analyses. In each case, the 600 to 1,000 base pairs of phi X174 DNA (derived from different regions of the phi X174 genome) were present as single inserts, located in either the early or late SV40 regions; the deletion of SV40 DNA was greater than the size of the insert; and the remaining portions of the hybrid genome were indistinguishable from wild-type SV40 DNA, as judged by both mapping and biological tests. Hence, apart from the deletion which accommodates the phi X174 DNA insert, no other rearrangements of SV40 DNA were detected. The restriction map of a SV40-phi X174 recombinant DNA isolate before molecular cloning was indistinguishable from those of two separate cloned derivatives of that isolate, indicating that the species cloned was the major amplifiable recombinant structure generated by a single recombinant-producing cell. The relative simplicity of the SV40-phi X174 recombinant DNA examined is consistent with the notion that most recombinant-producing BSC-1 cells support single recombination events generating only one amplifiable recombinant structure.     

Studies of the recognition sequence of phi X174 gene A protein. Cleavage site of phi X gene A protein in St-1 RFI DNA.

It is already known that phi X gene A protein converts besides phi X RFI DNA also the RFI DNAs of the single-stranded bacteriophages G4, St-1, alpha 3 and phi K into RFII DNA. We have extended this observations for bacteriophages G14 and U3. Restriction enzyme analysis placed the phi X gene A protein cleavage site in St-1 RF DNA in the HinfI restriction DNA fragment F10 and in the overlapping HaeIII restriction DNA fragment Z7. The exact position and the nucleotide sequence at the 3'-OH end of the nick were determined by DNA sequence analysis of the single-stranded DNA subfragment of the nicked DNA fragment F10 obtained by gelelectrophoresis in denaturing conditions. A stretch of 85 nucleotides of St-1 DNA around the position of the phi X gene A protein cleavage site was established by DNA sequence analysis of the restriction DNA fragment Z7F1. Comparison of this nucleotide sequence with the previously determined nucleotide sequence around the cleavage site of phi X gene A protein in phi X174 RF DNA and G4 RF DNA revealed an identical sequence of only 10 nucleotides. The results suggest that the recognition sequence of the phi X174 gene A protein lies within these 10 nucleotides.     

recA (Srf) suppression of recF deficiency in the postreplication repair of UV-irradiated Escherichia coli K-12.

The mechanism by which recA (Srf) mutations (recA2020 and recA801) suppress the deficiency in postreplication repair shown by recF mutants of Escherichia coli was studied in UV-irradiated uvrB and uvrA recB recC sbcB cells. The recA (Srf) mutations partially suppressed the UV radiation sensitivity of uvrB recF, uvrB recF recB, and uvrA recB recC sbcB recF cells, and they partially restored the ability of uvrB recF and uvrA recB recC sbcB recF cells to repair DNA daughter-strand gaps. In addition, the recA (Srf) mutations suppressed the recF deficiency in the repair of DNA double-strand breaks in UV-irradiated uvrA recB recC sbcB recF cells. The recA2020 and recA801 mutations do not appear to affect the synthesis of UV radiation-induced proteins, nor do they appear to produce an altered RecA protein, as detected by two-dimensional gel electrophoresis. These results are consistent with the suggestion (M. R. Volkert and M. A. Hartke, J. Bacteriol. 157:498-506, 1984) that the recA (Srf) mutations do not act by affecting the induction of SOS responses; rather, they allow the RecA protein to participate in the recF-dependent postreplication repair processes without the need of the RecF protein.     

Selectivity of the excision of alkylation products in a xeroderma pigmentosum-derived lymphoblastoid line.

Lymphoblastoid cell derived from a complementation group C xeroderma patient were unable to remove 06-methyl guanine residues formed in DNA by treatment of cells with low concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The xeroderma cells were competent in their ability to excise 3-methyl adenine adducts. MNNG treatment induced excision repair in the xeroderma line and in addition the treatment resulted in the presence of numerous single-strand breaks in the DNA. The single gene, UV-excision-defective mutants of Escherichia coli, uvrA and uvrB, are able to excise MNNG-induced 06-methyl guanine adducts indicating that excision of this compound is not due to operation of UV endonuclease system.