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1.
Mutagenesis ; 28(3): 341-50, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23446177

RESUMO

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.


Assuntos
Alquilantes/toxicidade , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/fisiologia , Mutagênese/efeitos dos fármacos , Mutagênicos/toxicidade , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Técnicas de Inativação de Genes , Ordem dos Genes , Teste de Complementação Genética , Mutação , O(6)-Metilguanina-DNA Metiltransferase/genética , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Plasmídeos/genética
2.
Genes Environ ; 41: 11, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30988834

RESUMO

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.

3.
J Med Chem ; 51(1): 159-66, 2008 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-18067241

RESUMO

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). Steady-state kinetics analysis of the incorporation of these triphosphate analogues by a poliovirus RdRp, 3D (pol), revealed that while the incorporation efficiency of 9a was comparable to RTP, 9b and 9c showed lower efficiency than RTP. Antipolioviral activity of 7a and 7b was much more moderate than ribavirin, and 7c showed no antipolioviral activity. Effects of substituting groups on the incorporation efficiency by 3D (pol) and a strategy for a rational design of more active ribavirin analogues are discussed.


Assuntos
Antivirais/síntese química , Organofosfatos/síntese química , Poliovirus/efeitos dos fármacos , Poliovirus/genética , RNA Viral/genética , Ribavirina/análogos & derivados , Ribavirina/síntese química , Amidas/síntese química , Amidas/química , Antivirais/química , Antivirais/farmacologia , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Mutação , Organofosfatos/química , Organofosfatos/farmacologia , Poliovirus/enzimologia , RNA Polimerase Dependente de RNA/química , Ribavirina/química , Ribavirina/farmacologia , Relação Estrutura-Atividade
4.
Mutagenesis ; 23(6): 509-13, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18765421

RESUMO

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. However, the lesion in 5'-CCG(o)-3' can cause efficient G-to-T transversion in a strain lacking the rad30 gene which encodes yeast DNA polymerase eta (Ypoleta). To explore the properties associated with this translesion synthesis (TLS), the same two oligonucleotides possessing an 8-oxoG were used as templates for a standing-start primer extension assay, and the nucleotide incorporation opposite 8-oxoG was investigated. We found that dATP incorporation opposite 8-oxoG with Ypoleta was low for both sequences. In particular, very low dATP incorporation was observed for the 5'-CCG(o)-3' sequence. These results account for the efficient inhibition of mutagenesis by Ypoleta. TLS plays an important role in one DNA sequence in terms of avoiding mutagenesis induced by 8-oxoG in yeast. In contrast, human yeast DNA polymerase eta showed higher dATP incorporation rates even with the 5'-CCG(o)-3' sequence.


Assuntos
Guanina/análogos & derivados , Mutagênese , Saccharomyces cerevisiae/efeitos dos fármacos , Sequência de Bases , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Guanina/toxicidade , Dados de Sequência Molecular , Mutação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo
5.
Mutat Res ; 640(1-2): 107-12, 2008 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-18243250

RESUMO

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. coli. We find that while methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, this genetic background does not affect mutagenicity by ethylating agents. Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis. To further understand this difference an early step of repair was examined with these alkylating agents. A comparison of binding affinities of MutS with O(6)-alkylated guanine base paired with thymine, which could lead to transition mutations, versus cytosine which could not, was tested. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O(6)-MeG:T, O(6)-MeG:C, O(6)-EtG:T, O(6)-EtG:C, G:T and G:C. Dissociation constants (K(d)), which reflect the strength of binding, followed the order G:T->O(6)-MeG:T->O(6)-EtG:T-=O(6)-EtG:C-> or =O(6)-MeG:C->G:C. These results suggest that a thymine base paired with O(6)-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O(6)-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induced lesions than those caused by ethylation.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Mutação , Oligonucleotídeos/metabolismo , Alquilantes/farmacologia , Alquilação , Sequência de Bases , Frequência do Gene , Guanina/metabolismo , Dados de Sequência Molecular
6.
Nucleic Acids Res ; 34(22): 6438-49, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17130163

RESUMO

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.


Assuntos
Antirretrovirais/química , Citidina/análogos & derivados , Mutagênese , Transcrição Reversa , Ribonucleotídeos/química , Citidina/química , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Transcriptase Reversa do HIV/metabolismo , Células HeLa , Humanos , Cinética , RNA Polimerase II/metabolismo , Retroviridae/genética , Moldes Genéticos
7.
Artigo em Inglês | MEDLINE | ID: mdl-28283089

RESUMO

Alkylating agents are known to induce the formation of O6-alkylguanine (O6-alkG) and O4-alkylthymine (O4-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 O6-methylguanine more efficiently than the suppression of GC-to-AT mutations caused by O6-ethylguanine. However, the manner by which O4-alkyT lesions are repaired remains unclear. In the present study, we investigated the repair pathway involved in the repair of O4-alkT. The E. coli CC106 strain, which harbors Δprolac in its genomic DNA and carries the F'CC106 episome, can be used to detect AT-to-GC reverse-mutation of the gene encoding ß-galactosidase. Such AT-to-GC mutations should be induced through the formation of O4-alkT at AT base pairs. As expected, an O6-alkylguanine-DNA alkyltransferase (AGT) -deficient CC106 strain, which is defective in both ada and agt genes, exhibited elevated mutant frequencies in the presence of methylating agents and ethylating agents. However, in the UvrA-deficient strain, the methylating agents were less mutagenic than in wild-type, while ethylating agents were more mutagenic than in wild-type, as observed with agents that induce O6-alkylguanine modifications. Unexpectedly, the mutant frequencies decreased in a MutS-deficient strain, and a similar tendency was observed in MutL- or MutH-deficient strains. Thus, MMR appears to promote mutation at AT base pairs. Similar results were obtained in experiments employing double-mutant strains harboring defects in both MMR and AGT, or MMR and NER. E. coli MMR enhances AT-to-GC mutagenesis, such as that caused by O4-alkylthymine. We hypothesize that the MutS protein recognizes the O4-alkT:A base pair more efficiently than O4-alkT:G. Such a distinction would result in misincorporation of G at the O4-alkT site, followed by higher mutation frequencies in wild-type cells, which have MutS protein, compared to MMR-deficient strains.


Assuntos
Alquilantes/farmacologia , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Escherichia coli/genética , Pareamento de Bases , Mutação
8.
Nucleic Acids Res ; 30(23): 5129-35, 2002 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-12466536

RESUMO

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. Deletion of REV1 decreased the transformation efficiency and the incorporation of cytosine nearly to a background level. In contrast, deletion of RAD30 did not affect them. We compared the mutagenic specificity with that of a tetrahydrofuran abasic site (F), an abasic analog used widely. Its mutation spectrum was clearly different from that of O. Adenine, not cytosine, was most favorably incorporated. However, deletion of REV1 decreased the transformation efficiency with F-containing oligonucleotide as in the case of O. These results suggest that the bypass mechanism of F is different from that of O, although the bypasses in both cases are dependent on REV1. We also found that the mutagenic specificity of F can be affected by not only the adjacent bases, but also a base located two positions away from F.


Assuntos
DNA/química , Furanos/química , Mutagênese , Saccharomyces cerevisiae/genética , Modelos Genéticos , Mutação , Oligonucleotídeos/química , Oligonucleotídeos/genética , Transformação Genética , Uracila/química
9.
Genetics ; 161(4): 1363-71, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12196386

RESUMO

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. T --> G. C transition mutations. We have found that the Escherichia coli DNA mismatch-repair system can protect cells against this mutagenic action. At a low dose, dP is much more mutagenic in mismatch-repair-defective mutH, mutL, and mutS strains than in a wild-type strain. At higher doses, the difference between the wild-type and the mutator strains becomes small, indicative of saturation of mismatch repair. Introduction of a plasmid containing the E. coli mutL(+) gene significantly reduces dP-induced mutagenesis. Together, the results indicate that the mismatch-repair system can remove dP-induced replication errors, but that its capacity to remove dP-containing mismatches can readily be saturated. When cells are cultured at high dP concentration, mutant frequencies reach exceptionally high levels and viable cell counts are reduced. The observations are consistent with a hypothesis in which dP-induced cell killing and growth impairment result from excess mutations (error catastrophe), as previously observed spontaneously in proofreading-deficient mutD (dnaQ) strains.


Assuntos
Pareamento Incorreto de Bases , Reparo do DNA/fisiologia , Escherichia coli/genética , Desoxicitidina/genética , Escherichia coli/fisiologia , Mutagênese
10.
Mutat Res ; 578(1-2): 79-87, 2005 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-15896814

RESUMO

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. A point mutation in the BRCT domain is known to abolish the second function. In the present research, we have studied the effects of deletion of the BRCT domain and a point mutation at the two amino acids in the putative polymerase active center. We have introduced an abasic site, its tetrahydrofuran analog, and a 6-4 thymine-thymine photoproduct using the oligonucleotide transformation assay. Translesion efficiencies were estimated from the transforming activities of the oligonucleotides with a lesion, and the mutation spectra were analyzed by DNA sequencing of the transformants. Results showed that the lack of the BRCT domain reduced translesion efficiencies, but that substantial translesion synthesis took place. The mutation spectra of the lesions were not greatly affected. Therefore, the BRCT domain may be important, but dispensable for translesion synthesis. In contrast, the polymerase mutation, rev1AA, has only small effects on the translesion efficiencies, but the mutation spectra were greatly affected; the incorporation of dCMP opposite the lesions was specifically lost. This clearly shows that the polymerase domain is responsible for the dCMP incorporation. The effect of Poleta was also analyzed. From all the results DNA polymerases other than these two translesion polymerases, too, seem to initiate the translesion synthesis.


Assuntos
Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , DNA/biossíntese , Análise Mutacional de DNA , DNA Fúngico , Modelos Biológicos , Nucleotidiltransferases/genética , Mutação Puntual , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência
11.
Mutat Res ; 502(1-2): 53-60, 2002 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-11996972

RESUMO

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. It is supposed to be a miscoding, but otherwise good template for DNA polymerases. The other lesion was the TT pyrimidine(6-4)pyrimidone photoproduct, one of the typical UV lesions, which blocks DNA replication. These oligonucleotides were used to transform yeast cyc1 mutants with ochre nonsense mutation to Cyc1+. As expected from its templating properties in vitro, the transforming activity of dP-containing oligonucleotides was similar to those of unmodified oligonucleotides. Results indicated that dP may direct incorporation of guanine and adenine at a ratio of 1:20 or more in vivo. An oligonucleotide containing the photoproduct showed the transforming activity of as low as 3-5% of that of the corresponding unmodified oligonucleotide. This bypass absolutely required REV1 gene. The sequence analysis of the transformants has shown that the lesion was read as TT and TC at a ratio of 3:7, indicating its high mutagenic potential.


Assuntos
Dano ao DNA , DNA Fúngico/genética , Mutagênicos/farmacologia , Oligonucleotídeos/farmacologia , Saccharomyces cerevisiae/genética , Sequência de Bases , Primers do DNA
12.
Nucleic Acids Symp Ser (Oxf) ; (53): 217-8, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19749338

RESUMO

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.


Assuntos
Citosina/química , DNA/química , Compostos de Amônio Quaternário/química , Sulfitos/química , Uridina/química , Cinética
13.
Curr Protoc Nucleic Acid Chem ; Chapter 6: Unit 6.10, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18551428

RESUMO

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.


Assuntos
Metilação de DNA , Análise de Sequência de DNA/métodos , Sulfitos/química , 5-Metilcitosina/química , Animais , Composição de Bases , Sequência de Bases , DNA , Desaminação , Genoma/genética , Dados de Sequência Molecular , Solubilidade , Soluções
14.
Nucleic Acids Symp Ser (Oxf) ; (52): 531-2, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18776488

RESUMO

Translesion synthesis (TLS), an important mechanism in cells refers to bypassing the DNA damage blockage on replication fork. Yeast TLS polymerase eta (poleta) is able to bypass 7,8-dihydro-8-oxoguanine (8-oxoG) on DNA with high fidelity by incorporation of dCTP opposite 8-oxoG rather than dATP to avoid G to T transversion mutation. We have shown the 5' nearest base next to 8-oxoG affects the G to T mutation by yeast and human poleta previously. In this study, the insertion efficiency of dCTP opposite 8-oxoG in various DNA sequences was kinetically investigated using yeast poleta. Based on K(m) and V(max), we demonstrated that the insertion efficiencies were also influenced by the 5' neighboring nucleotide next to 8-oxoG. The lowest V(max)/K(m) was observed when cytosine was 5' neighbouring base to 8-oxoG, in agreement with previous results in which dCTP incorporation to 8-oxoG was lowest when cytosine is on the 5'-side next to the lesion.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxicitosina/metabolismo , Guanina/análogos & derivados , DNA/biossíntese , DNA/química , Guanina/química , Cinética , Leveduras/enzimologia
15.
Nucleic Acids Symp Ser (Oxf) ; (51): 47-8, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18029579

RESUMO

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.


Assuntos
5-Metilcitosina/química , Metilação de DNA , Compostos de Amônio Quaternário/química , Análise de Sequência de DNA , Sulfitos/química , Linhagem Celular Tumoral , Citosina/química , Desaminação , Genômica , Humanos , Ureia/química
16.
Nucleic Acids Symp Ser (Oxf) ; (51): 49-50, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18029580

RESUMO

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.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Guanina/análogos & derivados , Sequência de Bases , Dano ao DNA , Guanina/química , Humanos , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Moldes Genéticos
17.
Nucleic Acids Symp Ser (Oxf) ; (51): 211-2, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18029661

RESUMO

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. Oligonucleotides containing 2-hydroxyadenine or 5-hydroxycytosine were used for the transformation. The oligonucleotides showed transforming activities similar to unmodified oligonucleotides. This indicates that no repair systems were working on them. The sequencing data of the transformants showed that 5-hydroxycytosine and 2-hydroxyadenine are read mainly as cytosine and adenine. We will also discuss the mechanism of oligonucleotide transformation and its application to the mutagenesis study.


Assuntos
Citosina/análogos & derivados , Dano ao DNA , Guanina/química , Mutagênese , Oligonucleotídeos/química , Citosina/química , Análise Mutacional de DNA , Saccharomyces cerevisiae/genética , Transformação Genética
18.
Nucleic Acids Symp Ser (Oxf) ; (50): 69-70, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17150821

RESUMO

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.


Assuntos
Citosina/química , Metilação de DNA , Análise de Sequência de DNA/métodos , Sulfitos/química , Ureia/química , 5-Metilcitosina/química , Linhagem Celular Tumoral , DNA/química , Desaminação , Humanos , Reação em Cadeia da Polimerase
19.
Nucleic Acids Symp Ser (Oxf) ; (49): 97-8, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-17150651

RESUMO

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.


Assuntos
Citosina/análogos & derivados , Mutação , Oxazinas/química , Pirimidinas/química , DNA Polimerase Dirigida por RNA/metabolismo , Transcrição Reversa , Vírus da Mieloblastose Aviária/enzimologia , Pareamento de Bases , Cromatografia Líquida de Alta Pressão , Citosina/química , HIV/enzimologia
20.
Yeast ; 22(11): 895-906, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16134092

RESUMO

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.


Assuntos
Galactose/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Supressão Genética , Transativadores/genética , Fatores de Transcrição/fisiologia , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Complexo Mediador , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica
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