Chapter 21 : DNA Methylation and Mismatch Repair

Abstract:

Sucrose gradient sedimentation of F-lac is used to demonstrate physical transfer of the element from donor to recipient at the non-permissive temperature. Little was known about DNA methylation at that time (1971) other than that 6-methyladenine (6-meA) and 5-methylcytosine (5-meC) were present in E. coli DNA. Pseudomonas cells were mutagenized with ethyl methanesulfonate and incorporated in a soft agar layer on minimal agar lacking isoleucine and valine but containing a high level of ampicillin. The best approach to isolate mutants deficient in methylation was decided. The procedure used was based on two prior observations. First, it was known that DNA isolated from E. coli grown in the presence of ethionine, a methionine analog, was deficient in methylation because it was a substrate for the transfer of methyl groups from S-adenosyl-methionine (SAM) to DNA in crude extracts. Second, Herb Boyer’s lab had located the gene (near his) for cytosine methylation on the E. coli K-12 map by using this assay on recombinants obtained from crosses between K-12 and B, which does not have methylated cytosine in its DNA. The 14 DNA methylation mutants were grown with tritiated methionine and the amount of 6-meA and 5-meC was quantified. This led to the identification of three mutants lacking 6-meA and 11 lacking 5-meC. The isolation of mut suppressor strains was the author's entry into the DNA mismatch repair field. In the DNA repair world, N-methyl-N'- nitro-N-nitrosoguanidine (MNNG) is associated with base excision repair (BER) and cisplatin with nucleotide excision repair (NER).