Chloroethylating anticancer drug-induced mutagenesis and its repair in .

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.

E. coli mismatch repair enhances AT-to-GC mutagenesis caused by alkylating agents.

Alkylating agents are known to induce the formation of O-alkylguanine (O-alkG) and O-alkylthymine (O-alkT) in DNA. These lesions have been widely investigated as major sources of mutations. We previously showed that mismatch repair (MMR) facilitates the suppression of GC-to-AT mutations caused by O-methylguanine more efficiently than the suppression of GC-to-AT mutations caused by O-ethylguanine.

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.

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.

Influence of neighbouring base sequences on the mutagenesis induced by 7,8-dihydro-8-oxoguanine in yeast.

We have analysed the influence of neighbouring base sequences on the mutagenesis induced by 7,8-dihydro-8-oxoguanine (8-oxoG or G(o)), a typical oxidative lesion of DNA, using the yeast oligonucleotide transformation technique. Two oligonucleotides, oligo-CCG(o) and oligo-CGG(o), each possessing a single 8-oxoG residue and represented by the sequences 5'-CCG(o)-3' and 5'-CGG(o)-3', respectively, were introduced into a chromosome of Saccharomyces cerevisiae and their mutagenic potentials were compared. In a wild-type strain, 8-oxoG showed very weak mutagenic potential in both cases.

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.

Binding of MutS protein to oligonucleotides containing a methylated or an ethylated guanine residue, and correlation with mutation frequency.

The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of modified base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between recognition and the effects of methylating versus ethylating agents on mutagenesis, using a MutS-deficient strain of E.

Effects of introduction of hydrophobic group on ribavirin base on mutation induction and anti-RNA viral activity.

One of the possible mechanisms of antiviral action of ribavirin (1-beta- d-ribofuranosyl-1,2,4-triazole-3-carboxamide, 1) is the accumulation of mutations in viral genomic RNA. The ambiguous incorporation of 5'-triphosphate of ribavirin (RTP, 8) by a viral RNA-dependent RNA polymerase (RdRp) is a key step of the mutation induction. We synthesized three ribavirin analogues that possess hydrophobic groups, 4-iodo-1-beta- d-ribofuranosylpyrazole-3-carboxamide ( 7a), 4-propynyl-1-beta- d-ribofuranosylpyrazole-3-carboxamide ( 7b), and 4-phenylethynyl-1-beta-D-ribofuranosylpyrazole-3-carboxamide ( 7c), and the corresponding triphosphates ( 9a, 9b, and 9c, respectively).

Oligonucleotide transformation for the study of mutagenic specificities of DNA lesions in yeast.

We developed a method for the analyzing mutagenic potential of DNA damage based on the oligonucleotide transformation technique in yeast. Using this assay we have analyzed mutagenic specificities of various DNA lesions. In the present study, we analyzed the mutagenic properties of 2-hydroxyadenine and 5-hydroxycytosine in yeast.

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.

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.

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.

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.

UVA-induced degradation of 8-hydroxyguanine in oligonucleotide and the effect on its activities in yeast oligonucleotide transformation assay.

UVA-induced conversion of 8-hydroxyguanine in oligonucleotides was studied. By irradiation with 334 nm UVA light, 8-hydroxyguanine was completely changed to unknown compounds. Monomeric nucleoside may be much less labile to UVA.

Accelerated bisulfite-deamination of cytosine in the genomic sequencing procedure for DNA methylation analysis.

Understanding the biological consequences of DNA methylation is a current focus of intensive studies. A standard method for analyzing the methylation at position 5 of cytosines in genomic DNA involves chemical modification of the DNA with bisulfite, followed by PCR amplification and sequencing. Bisulfite deaminates cytosine, but it deaminates 5-methylcytosine only very slowly, thereby allowing determination of the methylated sites.

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.

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.

Roles of the polymerase and BRCT domains of Rev1 protein in translesion DNA synthesis in yeast in vivo.

Rev1p in yeast is essential for the translesion of abasic sites and 6-4 photoproducts. It plays a role as a translesion polymerase, but also supports translesion catalyzed by other polymerases. The protein has two domains, BRCT and Y-family polymerase.

The role of deoxycytidyl transferase activity of yeast Rev1 protein in the bypass of abasic sites.

We have studied the mutagenic specificity of abasic sites using the yeast oligonucleotides transformation assay. We introduced oligonucleotide containing a natural abasic site and a tetrahydrofuran abasic site into Rev1 mutants, rev1AA, which contains mutations of Asp467 and Glu468 residues of Rev1p to Ala in order to inactivate dCMP transferase activity, and rev1 delta, which lacks its whole coding sequence. The transformation efficiencies of rev1AA with abasic-containing oligonucleotides were lower than those of B7528, a strain proficient in REV1 gene, but much higher than rev1 delta mutant.

Difference between deoxyribose- and tetrahydrofuran-type abasic sites in the in vivo mutagenic responses in yeast.

We have analyzed the mutagenic specificity of an abasic site in DNA using the yeast oligonucleotide transformation assay. Oligonucleotides containing an abasic site or its analog were introduced into B7528 or its derivatives, and nucleotide incorporation opposite abasic sites was analyzed. Cytosine was most frequently incorporated opposite a natural abasic site (O) ('C-rule'), followed by thymine.

Saturation of DNA mismatch repair and error catastrophe by a base analogue in Escherichia coli.

Deoxyribosyl-dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is a potent mutagenic deoxycytidine-derived base analogue capable of pairing with both A and G, thereby causing G. C --> A. T and A.

Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo.

We have studied mutagenic specificities of DNA lesions in vivo in yeast CYC1 oligonucleotide transformation assay. We introduced two lesions into oligonucleotides. One was a nucleoside analog, 3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-7-one 2'-deoxyriboside (dP), which is highly mutagenic to bacteria.