RESUMEN
The efficiency of Xenopus laevis egg extract to repair T:G and A:C mismatched base pairs in unmethylated, hemimethylated and fullymethylated heteroduplexes was investigated. Filamentous phage M13mp18 and its derivative M13mp18/MP-1 (C changed to T inside the sequence dCC*C GGG, at the position 6248) were used for heteroduplexes construction. The three origins of mismatched base-pairs in the eukaryotic DNA are mimicked by in vitro methylation: hemimethylated DNA (me-/me+) for replication errors; unmethylated (me-/me-) and fully methylated DNA (me+/me+) for recombination heteroduplexes, and fullymethylated also for locally, spontaneously deaminated 5-methylcytosine (5meC) to T, generating the exclusively T:G mismatch. The methylations were in CpG dinucleotides, mostly characteristic ofeukaryotic cells [5, 24]. In vitro methylation was done by HpaII methylase which methylate central C of dCCGG sequence in the manner of eukaryotic methylation. The position of mismatched bases was chosen so that correction of mismatched bases in any strand would create the sequence for one of the "diagnostic" restriction endonucleases, either BstNI or MspI. Correction efficiency was about 10(8) repair events per egg equivalent. Correction in favor of C:G base pair restoration occurred regardless of the T:G or C:A mispairs, with almost equal efficiency. Repair of T:G to T:A was up to 10 times less efficient comparing to C:G, and repair of C:A to T:A was in our experimental system undetectable. No significant difference in repair efficiency of mismatched bases situated in unmethylated, hemimethylated or fullymethylated heteroduplexes indicate methylation-independent repair of mismatched bases in X. laevis oocite extracts.
Asunto(s)
Disparidad de Par Base , Metilación de ADN , Reparación del ADN , Óvulo/metabolismo , Animales , Islas de CpG , ADN-Citosina Metilasas/metabolismo , Análisis Heterodúplex , Plásmidos/metabolismo , XenopusRESUMEN
It has been established that very short patch (VSP) mismatch repair, depending in Escherichia coli on MutL, MutS and Dcm functions, is responsible for the hyper-recombinogenic effect of a class of genetic markers. We show that VSP repair requires the presence of the complete DNA polymerase I enzyme. The absence of endonuclease activities involved in the repair of base-loss sites, Nth, Nfo and Xth, does not affect VSP repair. Implications for the mechanism of the VSP repair are discussed.
Asunto(s)
ADN Polimerasa I/metabolismo , Reparación del ADN , Escherichia coli/genética , Composición de Base , Secuencia de Bases , Cruzamientos Genéticos , Escherichia coli/metabolismo , Marcadores Genéticos , Mutación , Recombinación GenéticaRESUMEN
Unmethylated DNA heteroduplexes with a large single stranded loop in one strand have been prepared from separated strands of DNA from two different strains of bacteriophage lambda, one of which has a approximately 800 base pair IS1 insertion in the cI gene. The results of transfections with these heteroduplexes into wild-type and mismatch repair deficient bacteria indicate that such large non-homologies are not repaired by the Escherichia coli mismatch repair system. However, the results do suggest that some process can act to repair such large non-homologies in heteroduplex DNA. Transfections of a series of recombination and excision repair deficient mutants suggest that known excision or recombination repair systems of E. coli are not responsible for the repair. Repair of large non-homologies may play a role in gene conversion involving large insertion or deletion mutations.
Asunto(s)
ADN Bacteriano/genética , Ácidos Nucleicos Heterodúplex/genética , Composición de Base , Secuencia de Bases , Colifagos/genética , Reparación del ADN , Mutación , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
The relationship between survival and mutagenesis in Escherichia coli after fractionated ultraviolet (UV) irradiation was studied. The cells were incubated either in buffer or nutrient media. Regardless of incubation conditions, greater survival is observed after fractionated irradiation than after acute irradiation. When the cells are incubated in buffer, UV mutagenesis decreases with an increase in the number of dose fractions. However, when the cells are cultivated in nutrient media, the increased survival (i.e., the enhanced capacity for repair) is coupled with the enhanced capacity for UV mutagenesis. We, therefore, assume that during incubation in nutrient media, fractionated irradiation leads to full and prolonged expression of all UV-inducible (SOS) genes, including those required for mutagenesis.