Chloroethylating anticancer drug-induced mutagenesis and its repair in Escherichia coli.

BACKGROUND: Chloroethylnitrosourea (CENU) derivatives, such as nimustine (ACNU) and carmustine (BCNU), are employed in brain tumor chemotherapy due to their ability to cross the blood-brain barrier. They are thought to suppress tumor development through DNA chloroethylation, followed by the formation of interstrand cross-links (ICLs) that efficiently block replication and transcription. However, the alkylation of DNA and ICLs may trigger genotoxicity, leading to tumor formation as a side effect of the chemotherapeutic treatment. Although the involvement of O 6-alkylguanine-DNA alkyltransferase (AGT) in repairing chloroethylated guanine (O 6-chloroethylguanine) has been reported, the exact lesion responsible for the genotoxicity and the pathway responsible for repairing it remains unclear. RESULTS: We examined the mutations induced by ACNU and BCNU using a series of Escherichia coli strains, CC101 to CC111, in which reverse mutations due to each episome from F'101 to F'106 and frameshift mutations due to each episome from F'107 to F'111 could be detected. The mutant frequency increased in E. coli CC102, which can detect a GC to AT mutation. To determine the pathway responsible for repairing the CENU-induced lesions, we compared the frequency of mutations induced by CENU in the wild-type strain to those in the ada, ogt (AGT-deficient) strain, uvrA (nucleotide excision repair (NER)-deficient) strain, mismatch repair (MMR)-deficient strains, and recA (recombination deficient) strain of E. coli CC102. The frequencies of mutations induced by ACNU and BCNU increased in the ada, ogt strain, demonstrating that O 6-chloroethylguanines were formed, and that a portion was repaired by AGT.Mutation induced by ACNU in NER-deficient strain showed a similar profile to that in AGT-deficient strain, suggesting that an NER and AGT play at the similar efficacy to protect E. coli from mutation induced by ACNU. O 6-Chloroethylguanine is reported to form ICLs if it is not repaired. We examined the survival rates and the frequencies of mutations induced by ACNU and BCNU in the uvrA strain, the recA strain, as well as a double-deficient strain of CC102. The mutation profile of the double-deficient strain was similar to that of the NER-deficient strain, suggesting that an NER protects E. coli from mutations but not recombination. In addition, cell death was more pronounced in the uvrA, recA double-deficient strain than in the single-deficient strains. CONCLUSION: These results suggest that the toxic lesions induced by CENU were repaired additively or synergistically by NER and recombination. In other words, lesions, such as ICLs, appear to be repaired by NER and recombination independently.

Distinct pathways for repairing mutagenic lesions induced by methylating and ethylating agents.

DNA alkylation damage can be repaired by nucleotide excision repair (NER), base excision repair (BER) or by direct removal of alkyl groups from modified bases by O(6)-alkylguanine DNA alkyltransferase (AGT; E.C. 2.1.1.63). DNA mismatch repair (MMR) is also likely involved in this repair. We have investigated alkylation-induced mutagenesis in a series of NER- or AGT-deficient Escherichia coli strains, alone or in combination with defects in the MutS, MutL or MutH components of MMR. All strains used contained the F'prolac from strain CC102 (F'CC102) episome capable of detecting specifically lac GC to AT reverse mutations resulting from O(6)-alkylguanine. The results showed the repair of O(6)-methylguanine to be performed by AGT ≫ MMR > NER in order of importance, whereas the repair of O(6)-ethylguanine followed the order NER > AGT > MMR. Studies with double mutants showed that in the absence of AGT or NER repair pathways, the lack of MutS protein generally increased mutant frequencies for both methylating and ethylating agents, suggesting a repair or mutation avoidance role for this protein. However, lack of MutL or MutH protein did not increase alkylation-induced mutagenesis under these conditions and, in fact, reduced mutagenesis by the N-alkyl-N-nitrosoureas MNU and ENU. The combined results suggest that little or no alkylation damage is actually corrected by the mutHLS MMR system; instead, an as yet unspecified interaction of MutS protein with alkylated DNA may promote the involvement of a repair system other than MMR to avoid a mutagenic outcome. Furthermore, both mutagenic and antimutagenic effects of MMR were detected, revealing a dual function of the MMR system in alkylation-exposed cells.

Progress in the bisulfite modification of nucleic acids.

Bisulfite modification is a principal tool for analyzing DNA methylation, the methyl substitution at position 5 of cytosine residues. Hypermethylation is known to cause silencing of genes, which may result in cell function failures. DNA methylation analysis is therefore a focus of attention in various fields of biological sciences, including even clinical practices for treatment of cancer patients. In 2004, we reported that the bisulfite modification of DNA necessary in this analysis can be speeded up significantly by using a high concentration ammonium bisulfite solution (10 M), in place of traditional sodium bisulfite solution of 5 M concentration. Evaluations on this newer protocol have now come out from several laboratories, showing that this quick process can yield results with greater accuracy compared to those obtainable with widely-practiced low-concentration methods. Another aspect reported here is a study on the desulfonation of uracil-bisulfite adduct to form uracil, the last step of the bisulfite-conversion of cytosine to uracil. Kinetic measurements for the desulfonation of uridine-bisulfite adduct at a near-neutral pH region are described.

Bisulfite modification for analysis of DNA methylation.

Bisulfite is known to deaminate cytosine in nucleic acids, while 5-methylcytosine resists this bisulfite action. For this reason, bisulfite treatment has been used for detecting 5-methylcytosine in DNA, a minor component of eukaryotic DNA, presently recognized as playing an important role in the control of gene function. This procedure, called bisulfite genomic sequencing, is a principal method for the analysis of DNA methylation in various biological phenomena, including human diseases such as cancer. This unit describes an efficient procedure utilizing a newly developed high-concentration bisulfite solution. Protocols for this methodology are supplemented with discussions focused on chemical aspects of the bisulfite treatment.

Chemistry of bisulfite genomic sequencing; advances and issues.

Methylation at position 5 of cytosine in DNA plays a major role in epigenetic gene control. The methylation analysis can be performed by bisulfite genomic sequencing. Conventional procedures in this analysis include a treatment of single stranded DNA with 3-5 M sodium bisulfite at pH 5 and at 50-55 degrees for 4-20 hr. This will convert cytosine into uracil, while 5-methylcytosine resists this deamination. Amplification by PCR of the bisulfite-treated DNA followed by sequencing reveals the positions of 5-methylcytosine in the gene. We reported recently that the whole procedure can be speeded up by use of a highly concentrated bisulfite solution, 10 M ammonium bisulfite. We also reported that urea, which has been often added to the reaction mixture with the purpose of facilitating the reaction, may not work as anticipated. This time, we would like to address the need for further investigating the chemistry of the bisulfite modification of DNA. Particularly important is to study side reactions that may occur due to the exhaustive bisulfite treatment required for achieving complete deamination of all the cytosine residues in a given sample of DNA.

Nucleotide incorporation against 7,8-dihydro-8-oxoguanine is influenced by neighboring base sequences in TLS DNA polymerase reaction.

7,8-Dihydro-8-oxoguanine (8-oxoG) is a well-known oxidative lesion in DNA and is related to carcinogenesis and ageing processes. Misincorporation of dATP opposite to 8-oxoG leads to G --> T transversion mutations. DNA sequence has been proved as an important factor influencing the replication and enzymatic repair of various types of damages. To explore the influence of sequence effect on the properties of translesion synthesis (TLS) polymerase bypass of 8-oxoG, oligonucleotides with an 8-oxoG in different sequence contexts were used. We conclude that the 5'-nearest base next to 8-oxoG has significant effects in the G --> T mutation by hpoleta.

Does urea promote the bisulfite-mediated deamination of cytosine in DNA? Investigation aiming at speeding-up the procedure for DNA methylation analysis.

Methylation of cytosine in DNA at position 5 plays important roles in gene functions. Changes in the methylation status are linked to cancer. These studies have been developed on the basis of determining 5-methylcytosine residues [mC] in DNA. This analytical procedure uses the principle that bisulfite deaminates cytosine [C] but it deaminates mC only very slowly. Thus, 'bisulfite genomic sequencing' involves treatment of a given DNA sample with bisulfite followed by PCR amplification and sequencing, through which C residues in the original DNA are found as T and mC as C. In this procedure, a treatment with 3-5 M sodium bisulfite for 12-16 hr at 55 degrees C has been conventionally used. Recently, we were able to improve the efficiency of this procedure by introducing a highly concentrated (10 M) bisulfite solution. Aiming at further improvement of the procedure, we have now explored the effect of adding urea in this bisulfite treatment, as urea was reported to improve the deamination efficiency. Using 7.5 M ammonium bisulfite (pH 5.4) at 70 degrees C with or without the presence of 6 M urea, we performed deamination and sequencing of a DNA sample having known multiple CpG sites with mC. The deaminated DNAs were then subjected to PCR amplification followed by sequencing. In the 15 min-treated sample, the deamination extents were; C 96.5%, mC 1.1% for "bisulfite-only"; and C 90.3%, mC 1.4% for "bisulfite + urea". In the 30 min-treated sample, these values were; C 99.7%, mC 3.6% for "bisulfite only"; and C 99.7%, mC 2.1% for "bisulfite + urea". These results indicate that urea did not enhance the deamination efficiency. In the PCR, we did not observe significant improvements regarding the amounts of DNA necessary to obtain adequate amplification. Urea at 2 M, 4 M, and 8 M, showed no improvements. We conclude that urea gave no significant effect in the bisulfite genomic sequencing of the DNA used.

Template properties of mutagenic cytosine analogues in reverse transcription.

We have studied the mutagenic properties of ribonucleotide analogues by reverse transcription to understand their potential as antiretroviral agents by mutagenesis of the viral genome. The templating properties of nucleotide analogues including 6-(beta-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c](1,2)oxazin-7-one, N4-hydroxycytidine, N4-methoxycytidine, N4-methylcytidine and 4-semicarbazidocytidine, which have been reported to exhibit ambiguous base pairing properties, were examined. We have synthesized RNA templates using T3 RNA polymerase, and investigated the specificity of the incorporation of deoxyribonucleoside triphosphates opposite these cytidine analogues in RNA by HIV and AMV reverse transcriptases. Except for N4-methylcytidine, both enzymes incorporated both dAMP and dGMP opposite these analogues in RNA. This indicates that they would be highly mutagenic if present in viral RNA. To study the basis of the differences among the analogues in the incorporation ratios of dAMP to dGMP, we have carried out kinetic analysis of incorporation opposite the analogues at a defined position in RNA templates. In addition, we examined whether the triphosphates of these analogues were incorporated competitively into RNA by human RNA polymerase II. Our present data supports the view that these cytidine analogues are mutagenic when incorporated into RNA, and that they may therefore be considered as candidates for antiviral agents by causing mutations to the retroviral genome.

The Saccharomyces cerevisiae ESU1 gene, which is responsible for enhancement of termination suppression, corresponds to the 3'-terminal half of GAL11.

A DNA fragment enhancing efficiency of [PSI+]-dependent termination suppressor, sup111, was isolated from a genomic library of Saccharomyces cerevisiae and its function was attributed to an ORF of 1272 bp. This ORF, designated ESU1 (enhancer of termination suppression), corresponded to the 3'-terminal portion of GAL11. Contrasting to ESU1, GAL11 lowered the suppression efficiency of [PSI+] sup111. ESU1 possesses a TATA-like sequence of its own and three ATG codons following it within a distance of about 70 bp and all in the same reading frame as GAL11. A 52.7 kDa protein corresponding in size to the predicted Esu1 protein is detected by western blot analysis using anti-Gal11 antiserum. We therefore conclude that ESU1 is the gene that encodes a polypeptide corresponding to the C-terminal 424 amino acids of Gal11. It was further found that ESU1 increases the level of GAL11 mRNA and probably also of its own mRNA. Moreover, ESU1 increased the cellular level of mRNA transcribed from the leu2-1(UAA) mutant gene, while GAL11 did not. Based on these findings, we propose the following scheme for the events taking place in the [PSI+] sup111 cell that is transformed with an ESU1-bearing plasmid: (a) ESU1 stimulates transcription of leu2-1; (b) leu2-1 mRNA is not effectively degraded because of the possession of sup111, which belongs to the upf group; (c) [PSI+] causes increased mis-termination due to depletion of eRF3; (d) functional Leu2 product is made using leu2-1 mRNA; and (d) suppression of leu2-1 is eventually accomplished.

Mutagenic properties of ribonucleotide analogues in reverse transcription with HIV and AMV reverse transcriptases.

Pyrimidine analogues, N4-hydroxycytosine (C(oh)), N4-methoxycytosine (C(mo)) and 6H, 8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P) can form base pairs with both adenine and guanine. We examined the mutagenic properties of these ribonucleotide analogues in RNA in reverse transcription with HIV and AMV reverse transcriptases. Both reverse transcriptases incorporated dATP and dGTP opposite these analogues in RNA. The incorporation ratio of dGTP to dATP opposite each analogue was measured to estimate the potential for inducing U-to-C mutations. The potentials may be rC(oh) > rC(mo) > rP for both reverse transcriptases. It might be possible to induce mutations in the retroviral genomes and to develop a new antiviral therapy, if these analogues are incorporated by a human RNA polymerase.