Methylation of cytosines in DNA plays a variety of roles in nature. These include distinguishing """"""""self"""""""" DNA from foreign DNA, regulation of gene expression and tagging regions of sequence duplication. such uses of methylated cytosine appear to come with a drawback: methylated cytosines are known to be mutagenic. In both E. coli and in human cells, sites of cytosine methylation appear to be hotspots for C to T mutations. Spontaneous deamination of 5-methylcytosine (5meC) to thymine is thought to be the source of these mutations. Interestingly, E. coli and human cells also contain base mismatch correction systems that may play significant roles in reducing such mutations. Therefore, it is important to determine the rate of 5meC deamination in cells. It would be useful also to study the effect of various structural and physiological factors on the rate of 5-methylcytosine deamination, and the reduction in this rate achieved by the mismatch correction systems. A genetic system will be created in E. coli in which 5meC to T mutations can be positively selected. Kanamycin-resistant revertants arising as the result of 5meC to T transitions at Dcm sites [C5meC(A or T)GG] or at HpaII methylation sites (C5meCGG) will be quantitated. The physical state of the DNA will be varied by packaging it in phage capsids or by changing the sequence surrounding the 5meC. The effects of DNA replication, transcription and conjugation on the mutation rate will be studied. The ability of T/G mispairs resulting from 5meC deamination to be corrected under different conditions will be quantitated. HpaII methylation occurs in the same sequence context (CpG) as mammalian methylation. Sensitivity of eukaryotic cells to Kanamycin analogs such as G-418 and the ability of the kan gene to confer resistance to these analogs should allow extension of this genetic system to mammalian cells in the future.
