General methylation question - (Oct/11/2006 )
I'm a little confused and this may be a very dumb question but here goes. I'm trying to understand the process of how the sodium bisulfite modification works. As I understand it this is essentially the deamination of sulphonated cytosine (non-methylated) and subsequent desulphonation to uracil. However, due to steric hindrance or some other mechanism, methylated cytosines do not get converted.
What I'm a bit confused about is that CG suppression, which is responsible for the spontaneous deamination of 5'mc, appears to contradict this. How is it that deamination in the bisulfite modification affects only non-methlated cytosines whereas it only affects methylated cytosines in genome CG suppression? I would assume that it has something to do with the sulphonation step, but I don't know enough chemistry to really understand why that is.
Could anyone explain?
i think you have your answer in your post cancergeek.
My understanding is that you talking about two different chemistry reactions. Lets take CG suppression for example, spontaneous deamination of 5-methylcytosine gives rise to thymidine and is more difficult to correct by mismatch repair mechanisms. Deamination of cytosine results in uracil and this does occur spontaneously, but is readily repaired by the mismatch repair enzymes. The deamination processes are different. Bisulfite conversion is performed under acidic conditions and preferentially deaminates cytosine whilst the methyl group on 5-methylcytosine is protecting the amino group from deamination. Remember a hydrogen exists on the cytosine residue at position 5 where the methylgroup is on 5-methylcytosine.
It was something I had pondered once, a long time ago.