Incision at DNA G.T mispairs by extracts of mammalian cells occurs preferentially at cytosine methylation sites and is not targeted by a separate G.T binding reaction.
We have investigated the specificities of G.T mismatch binding proteins and of G.T mismatch cleavage in extracts of mammalian cells. G.T mismatch-specific protein:DNA complex formation by cell extracts was independent of the local sequence context of the mismatch. Cell extracts performed similar levels of protein binding to DNA substrates in which a single G.T mispair was preceded by T, G, A, C, or 5-meC. In contrast, incision by extracts of the T-containing strand of a G.T mismatch exhibited a strong sequence specificity and efficient strand cleavage was only observed when the mismatched G was in a CpG sequence. Thus, oligonucleotides containing either CpgGpT or 5meCpGGpT were efficiently incised, but not those containing GpGCpT, ApGTpT, or TpGApT sequences. Cell lines made resistant to the alkylating agent N-methyl-N-nitrosourea have previously been found to be defective in a G.T mismatch binding reaction. The defect in binding by extracts prepared from these cells extended to G.T mismatches in several sequence contexts. The variant extracts nevertheless incised G.T mismatches normally suggesting that this particular binding activity is not required for incision. The data indicate that incision by this activity is targeted to the CpG sequences in which G.T mismatches are formed by the mutagenic deamination of DNA 5-methylcytosine. In this regard the repair pathway resembles the very short patch (vsp) repair pathway in Escherichia coli.
Griffin
S
S
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, UK.
Karran
P
P
D044503:5-Methylcytosine; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship
D044503:5-Methylcytosine; D003596:Cytosine