Mismatch repair in xenopus egg extracts is not strand-directed by DNA methylation.
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.
Department of Molecular Genetics, Ruder Bosković Institute, Zagreb, Croatia. dina@rudjer.irb.hr
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Dzidić
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Radman
D000818:Animals; D020137:Base Pair Mismatch; D018899:CpG Islands; D019175:DNA Methylation; D004260:DNA Repair; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D020180:Heteroduplex Analysis; D010063:Ovum / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D014981:Xenopus
C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases