Kinetics of pyrimidine(6-4)pyrimidone photoproduct repair in Escherichia coli.

We compared the removal of pyrimidine(6-4)pyrimidone photoproducts [(6-4) photoproducts] and cyclobutane pyrimidine dimers (CPDs) from the genome of repair-proficient Escherichia coli, using monoclonal antibodies specific for each type of lesion. We found that (6-4) photoproducts were removed at a higher rate than CPDs in the first 30 min following a moderate UV dose (40 J/m2). The difference in rates was less than that typically reported for cultured mammalian cells, in which the removal of (6-4) photoproducts is far more rapid than that of CPDs.     

The role of the (6-4) photoproduct in ultraviolet light-induced transition mutations in E. coli

Available evidence rules out the possibility that cyclobutane dimers are the major premutagenic lesions responsible for point mutations at sites of adjacent pyrimidine residues in the experiment systems examined to date in sufficient detail, that is, UV-induced mutations in chromosome loci in E. coli and UV-induced mutations in the cI gene of phage lambda. However, it is likely that the major cytotoxic effects of UV irradiation can be attributed to the cyclobutane pyrimidine dimer, as these lesions occur at 10 times the frequency of other UV-induced photoproducts in the dose range of 0.1-100 J/m2. The evidence also suggests that cyclobutane pyrimidine dimers are the major lesions responsible for induction of the SOS response and that as such they play an important, though indirect role, in the formation of mutations in irradiated DNA. Cyclobutane dimers may also be the major lesions responsible for other types of UV-light-induced mutations such as deletions. None of the available evidence rules out (6-4) photoproducts as a major premutagenic lesion induced by UV irradiation using these experimental systems. On the contrary, the mutation spectrum induced both in the lacI gene and the cI gene of phage lambda is that predicted for mutations induced by (6-4) photoproducts. The observation that neither the premutagenic lesions nor the (6-4) photoproduct is subject to enzymatic photoreactivation also implies that the (6-4) photoproducts are premutagenic. As reviewed above, neither the photosensitization experiments nor the action spectrum of the (6-4) photoproducts rules out such a role. Might a lesion other than the (6-4) photoproduct be the major premutagenic lesion responsible for point mutations in these experimental systems? It cannot be ruled out that another as yet undefined minor photoproduct that occurs with the same sequence distribution specificity as that of the (6-4) photoproduct and that is also not subject to the reactivating treatments is more mutagenic than the (6-4) photoproduct itself. Candidates for such a lesion might include a photohydrate of the (6-4) photoproduct itself or as yet undefined photoproducts. However, we believe these alternative possibilities to be remote.(ABSTRACT TRUNCATED AT 400 WORDS)     

Escherichia coli DNA photolyase reverses cyclobutane pyrimidine dimers but not pyrimidine-pyrimidone (6-4) photoproducts

The effect of purified Escherichia coli DNA photolyase on the UV light-induced pyrimidine-pyrimidone (6-4) photoproduct and cyclobutane pyrimidine dimer was investigated in vitro using enzyme purified from cells carrying the cloned phr gene (map position, 15.7 min). Photoproducts were examined both as site-specific lesions in end-labeled DNA and as chromatographically identified products in uniformly labeled DNA. E. coli DNA photolyase removed cyclobutane dimers but had no activity on pyrimidine-pyrimidone (6-4) photoproducts. Photoreactivation can therefore be used to separate the biological effects of these two UV light-induced molecular lesions.     

Mutagenicity of a unique thymine-thymine dimer or thymine-thymine pyrimidine pyrimidone (6-4) photoproduct in mammalian cells.

The mutagenic properties of UV-induced photoproducts, both the cis-syn thymine-thymine dimer (TT) and the thymine-thymine pyrimidine pyrimidone (6-4) photoproduct [T(6-4)T] were studied in mammalian cells using shuttle vectors. A shuttle vector able to replicate in both mammalian cells and bacteria was produced in its single-stranded DNA form. A unique photoproduct was inserted at a single restriction site and after recircularization of the single-stranded DNA vector, this latter was transfected into simian COS7 cells. After DNA replication the vector was extracted from cells and used to transform bacteria. Amplified DNA was finally analyzed without any selective screening, DNA from randomly picked bacterial colonies being directly sequenced. Our results show clearly that both lesions are mutagenic, but at different levels. Mutation frequencies of 2 and 60% respectively were observed with the TT dimer and the T(6-4)T. With the TT dimer the mutations were targeted on the 3'-T. With the T(6-4)T a large variety of mutations were observed. A majority of G-->T transversions were semi-targeted to the base before the 5'-T of the photoproduct. These kinds of mutations were not observed when the same plasmid was transfected directly into SOS-induced JM105 bacteria or when the T(6-4)T oligonucleotide inserted in a different plasmid was replicated in SOS-induced SMH10 Escherichia coil bacteria. These semi-targeted mutations are therefore the specific result of bypass of the T(6-4)T lesion in COS7 cells by one of the eukaryotic DNA polymerases.     

The relative cytotoxicity and mutagenicity of cyclobutane pyrimidine dimers and (6-4) photoproducts in Escherichia coli cells

In order to calculate the relative cytotoxicity and mutagenicity of (5-6) cyclobutane pyrimidine dimers and (6-4) photoproducts, we have measured survival and mutation induction in UV-irradiated excision-deficient E. coli uvrA cells, with or without complete photoreactivation of the (5-6) dimers. Radioimmunoassays with specificity for (5-6) dimers or (6-4) photoproducts have shown that maximum photoreactivation eliminates all of the (5-6) dimers produced up to 10 Jm-2 254-nm light, while it has no effect on (6-4) photoproducts. These results were confirmed by measuring the frequency of T4 endonuclease V-sensitive sites. Based on the best fit equations for survival and mutation induction, we have found that the calculated cytotoxicity of (6-4) photoproducts is similar to that of (5-6) dimers; however, the former is much more mutagenic than the latter.     

HpaII methyltransferase is mutagenic in Escherichia coli.

A genetic reversion assay to study C-to-T mutations within CG sites in DNA is described. It was used to demonstrate that the presence of HpaII methyltransferase (MTase) in Escherichia coli causes a substantial increase in C-to-T mutations at CG sites. This is similar to the known mutagenic effects of E. coli MTase Dcm within its own recognition sequence. With this genetic system, a homolog of an E. coli DNA repair gene in Haemophilus parainfluenzae was tested for antimutagenic activity. Unexpectedly, the homolog was found to have little effect on the reversion frequency. The system was also used to show that HpaII and SssI MTases can convert cytosine to uracil in vitro. These studies define 5-methylcytosine as an intrinsic mutagen and further elaborate the mutagenic potential of cytosine MTases.     

Tryptophan H33 plays an important role in pyrimidine (6-4) pyrimidone photoproduct binding by a high-affinity antibody.

The importance of Trp H33 in antibody recognition of DNA containing a central pyrimidine (6-4) pyrimidone photoproduct was investigated. This residue was replaced by Tyr, Phe and Ala and the binding abilities of these mutants were determined by surface plasmon resonance and fluorescence spectroscopy. Conservative substitution of Trp H33 by Tyr or Phe resulted in moderate losses of binding affinity; however, replacement by Ala had a significantly larger impact. The fluorescence properties of DNA containing a (6-4) photoproduct were strongly affected by the identity of the H33 residue. DNA binding by both the wild-type and the W-H33-Y mutant was accompanied by a small degree of fluorescence quenching; by contrast, binding by the W-H33-F and W-H33-A mutants produced large fluorescence increases. Taken together, these variations in binding and fluorescence properties with the identity of the H33 residue are consistent with a role in photoproduct recognition by Trp H33 in the high-affinity antibody 64M5.     

Overexpression of vsr in Escherichia coli is mutagenic.

Overexpression of vsr in Escherichia coli stimulates transition and frameshift mutations. The pattern of mutations suggests that mutagenesis is due to saturation or inactivation of dam-directed mismatch repair.     

Complex formation of double-stranded DNA (6-4) photoproducts and anti-(6-4) photoproduct antibody Fabs.

DNA (6-4) photoproducts are major constituents of ultraviolet-damaged DNAs. We prepared double-stranded (ds) (6-4) DNA photoproducts and analyzed formation of their complexes with anti-(6-4) photoproduct antibody Fabs. Elution profiles of the mixtures of ds-(6-4) DNAs and Fabs from anion-exchange and gel-filtration columns indicate that Fab 64M-2 deprives 14mer ds-(6-4) DNA of single-stranded (ss) (6-4) DNA and shows no interaction with 18 mer ds-(6-4) DNA (A18). Fab 64M-5 with an approximately 100-fold higher affinity than Fab 64M-2 forms a complex with the ds-(6-4) DNA (A18), but partly dissociates another 18 mer ds-(6-4) DNA (A18-3), with a lowered G-C content, into ss-DNAs. From these results, antibody 64M-5 possibly accommodates the T(6-4)T photolesion moiety of the ds-(6-4) DNA (A18) by flipping out the moiety from its neighboring segments.     

The (6-4) photoproduct of thymine-thymine induces targeted substitution mutations in mammalian cells.

Two major ultraviolet-induced photolesions of TpT, a (6-4) photoproduct [T(6-4)T] and a cis-syn cyclobutane TT dimer (T=T), were incorporated into a predetermined site of one of the leading and lagging template strands of a double-stranded vector, and the modified DNAs were transfected into simian COS-7 cells. The DNAs replicated in the cells were recovered and were transfected again into Escherichia coli. The DNA replication efficiencies of plasmids containing T(6-4)T and T=T in the template strand for lagging strand synthesis were 93 and 79%, respectively, as compared with the unmodified DNA. Similar inhibitory effects were observed in the cases of the photoproducts in the template strand for leading strand synthesis (71 and 58%, respectively). These results indicated that T(6-4)T blocked DNA replication more weakly than T=T during leading and lagging strand syntheses in mammalian cells. The mutation frequencies of T(6-4)T were 2.3 and 4.7% in the leading and lagging template strands, respectively. The T=T lesion was less mutagenic and induced mutations with 0.2-0.7% frequencies. The T(6-4)T lesion primarily elicited 3'-T-->C substitutions, and T=T induced various types of mutations. These results indicate that T(6-4)T is more mutagenic than T=T during leading and lagging strand syntheses in simian cells. Moreover, this is the first evidence that shows T(6-4)T mainly elicits targeted substitutions at its 3'-T site in mammalian cells.     

(6-4) photoproducts and not cyclobutane pyrimidine dimers are the main UV-induced mutagenic lesions in Chinese hamster cells.

A partial revertant (RH1-26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1-26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1-26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1-26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1-26 cells but different from that of V-H1 cells. Since in RH1-26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.     

Chemical synthesis and properties of (6-4) photoproduct and its analogs.

We report the preparation of a deoxyribooligonucleotide containing a new thymine (6-4) photoproduct analog. The (6-4) photoproduct is one of the major forms of DNA lesions, and leads to mutation in DNA. An antibody (64M5) that binds the (6-4) photoproduct has been described. To investigate the interaction of the photoproduct with the 64M5 antibody, we prepared a (6-4) photoproduct analog in which the two thymines were connected with a formacetal linkage. With UV-irradiation, the thymine dimer with the formacetal linkage reacted to the (6-4) photoproduct faster than the phosphodiesterified dimer, and the yields of the analog was higher than those of the natural thymine dimer. The 64M5 antibody exhibited sufficient binding to a tetranucleotide containing the (6-4) photoproduct analog with a formacetal linkage, although the association constant was slightly lower than that for the natural lesion. This (6-4) photoproduct analog may be useful for investigation of other proteins that recognize the (6-4) photoproduct.     

Structural determination of the ultraviolet light-induced thymine-cytosine pyrimidine-pyrimidone (6-4) photoproduct.

Ultraviolet light induces damage to DNA, with the majority of the damage expressed as the formation of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts. The (6-4) photoproducts have been implicated as important UV light-induced premutagenic DNA lesions. The most abundant of the (6-4) products is the thymine-cytosine pyrimidine-pyrimidone (6-4) photoproduct, or TC (6-4) product. The structure of the TC (6-4) product was deduced by proton NMR, IR, and fast atom bombardment mass spectroscopy, and the product was found to differ from the previously described photoadduct, Thy(6-4)Pyo, by the presence of an amino group at the 5 position of the 5' pyrimidine. The implications of this structure on DNA base pairing and the induction of ultraviolet light-induced mutations are discussed.     

UV mutagenesis, cytotoxicity and split-dose recovery in a human-CHO cell hybrid having intermediate (6-4) photoproduct repair

Somatic cell hybrids constructed between UV-hypersensitive Chinese hamster ovary cell line UV20 and human lymphocytes were used to examine the influence of a human DNA repair gene, ERCC1, on UV photoproduct repair, mutability at several drug-resistance loci, UV cytotoxicity and UV split-dose recovery. In hybrid cell line 20HL21-4, which contains human chromosome 19, UV-induced mutagenesis at the APRT, HPRT and Na+/K+-ATPase loci was comparable to that in repair-proficient CHO AA8 cells, whereas cell line 20HL21-7, a reduced human-CHO hybrid not containing human chromosome 19, exhibited a hypermutable phenotype at all 3 loci indistinguishable from that of UV20 cells. The response of 20HL21-4 cells to UV cytotoxicity reflected substantial but incomplete restoration of wild-type UV cytotoxic response, whereas responses of UV20 and 20HL21-7 cell lines to UV cytotoxicity were essentially the same, reflecting several-fold UV hypersensitivity. Repair of UV-induced (5-6) cyclobutane dimers and (6-4) photoproducts was examined by radioimmunoassay; (6-4) photoproduct repair was deficient in UV20 and 20HL21-7 cell lines, and intermediate in 20HL21-4 cells relative to wild-type CHO AA8 cells. UV split-dose recovery in 20HL21-4 cells was also intermediate relative to AA8 cells. These results show that the human ERCC1 gene on chromosome 19 is responsible for substantial restoration of UV survival and mutation responses in repair-deficient UV20 cells, but only partially restores (6-4) UV photoproduct repair and UV split-dose recovery.     

Respective roles of pyrimidine dimer and pyrimidine (6-4) pyrimidone photoproducts in UV mutagenesis of simian virus 40 DNA in mammalian cells.

UV light induces DNA lesions which are mutagenic in mammalian cells. We used simian virus 40 tsB201 (unable to produce viral capsid at the restrictive temperature of 41 degrees C because of a point mutation in the VP1 gene) to analyze the mutagenic potency of the two major UV-induced lesions, pyrimidine dimers (Py-Py) and pyrimidine (6-4) pyrimidones [Py(6-4)Py], which are formed on the same nucleotide sites. The mutagenesis criterion was the reversion toward a wild-type growth phenotype. After UV irradiation (mainly at 254 nm), part of the DNA was treated with the photoreactivating enzyme of Escherichia coli, which monomerizes Py-Py but does not modify the Py(6-4)Py photoproduct. Higher survival and lower mutation frequency rates for the photoreactivated DNA indicated that the two lesions were lethal and mutagenic. The VP1 gene of some mutants was entirely sequenced. The mutation spectra showed that the two lesions did not induce the same mutation hot spots, although some sites were common to both. The induced mutation hot spots were not only correlated with lesion hot spots but seemed partially directed by local DNA structures.     

Expression of Met-(-1) angiogenin in Escherichia coli: conversion to the authentic less than Glu-1 protein

A method for obtaining authentic human angiogenin utilizing an Escherichia coli recombinant expression system is described. A synthetic gene encoding angiogenin was placed into a vector for direct expression under the control of a modified E. coli trp promoter. The protein was produced by the bacteria in an insoluble form and purified to homogeneity by cation-exchange and reversed-phase HPLC following reduction/solubilization and reoxidation. The protein isolated was identified as Met-(-1) angiogenin by amino acid analysis and tryptic peptide mapping; the latter demonstrated that all three disulfide bonds had formed correctly. Both the enzymatic and angiogenic activities of the Met-(-1) protein were equivalent to those of native angiogenin. A Met-(-1) Leu-30 derivative of angiogenin was also isolated and found to be fully active. Conversion of Met-(-1) angiogenin to the authentic less than Glu-1 protein was achieved by treatment with Aeromonas aminopeptidase under conditions in which the new N-terminal glutamine readily cyclizes nonenzymatically. This aminopeptidase treatment may have more general applicability for removal of undesirable N-terminal methionine residues from foreign proteins expressed in bacteria.     

In situ (6-4)photoproduct determination by laser cytometry and autoradiography

The UV-induced (6-4)photoproducts and their repair in individual human cells were quantitatively determined by argon-laser imaging microspectrofluorometry or autoradiography with a well-characterized monoclonal antibody against (6-4)photoproducts. (6-4)Photoproduct induction curves were linear as a function of UV dose, using both methods. The formation of (6-4)photoproducts was detected in the cells irradiated with as low as 10 and 25 J/m2 of UV by autoradiography and laser cytometry, respectively. Normal cells repaired more than 80% of the initial damage within 4 h post irradiation. In contrast, almost no repair was observed in xeroderma pigmentosum cells (complementation group A) within 8 h.     

Purification of a novel UV-damaged-DNA binding protein highly specific for (6-4) photoproduct.

UV damage-specific binding proteins are considered to play important roles in early responses of cells irradiated with UV, including damage recognition in the DNA repair process. We have surveyed nuclear and cytoplasmic proteins which bind selectively to UV-irradiated DNA using an electrophoretic mobility shift assay. We detected four distinct binding activities with different mobilities in fractions separated from HeLa cells by heparin chromatography. Three of them were found in nuclear extracts and one in cytoplasmic extracts. We purified one of the binding factors from nuclear extracts to homogeneity, which was designated NF-10 (the 10th fraction of nuclear extract on heparin chromatography). It migrated as a 40 kDa polypeptide in SDS-PAGE, and bound to UV-irradiated double- stranded DNA but not to unirradiated DNA. The binding pattern of the NF-10 protein to DNA irradiated with UV corresponded to the induction kinetics of (6-4) photoproduct. Removal of (6-4) photoproducts from UV- irradiated DNA by (6-4) photoproduct-specific photolyase diminished the binding of NF-10 protein. These results suggest that the NF-10 protein binds to UV-damaged DNA through (6-4) photoproduct. Immunoblot analysis using a monoclonal antibody revealed that the NF-10 protein was expressed in cell lines from all complementation groups of xeroderma pigmentosum, indicating that the NF-10 protein is a novel UV-damaged-DNA binding protein.     

Immunoprecipitation of pyrimidine(6-4)pyrimidone photoproducts and cyclobutane pyrimidine dimers in uv-irradiated DNA

Biological studies suggest that a significant proportion of the cytotoxicity observed in mammalian cells after uv irradiation may be due to damage other than cyclobutane dimers in DNA. Although pyrimidine-pyrimidone (6-4) photoproducts have been implicated as major contributors to cell lethality, their induction has been measured at considerably less than cyclobutane pyrimidine dimers when measured by chromatographic techniques. Because the yield of (6-4) photoproducts may be reduced by their lability to extreme heat and pH, we have advised an alternative, immunological quantification which does not require DNA hydrolysis. Affinity-purified rabbit antisera were used to precipitate low molecular weight 32P-labeled PM2 DNA irradiated with increasing fluences of uv light. DNA of known molecular weight was used to determine rates of induction for antibody-binding sites associated with (6-4) photoproducts and cyclobutane dimers. These rates were calculated to be 0.6 (6-4) photoproducts and 1.2 cyclobutane dimers/10(8) Da/J/m2. At low uv fluences (6-4) photoproducts were induced at one-half the rate of cyclobutane dimers, whereas at higher fluences (6-4) photoproducts predominated.