Mechanisms of cytosine deamination to uracil and repair of uracil:guanine mispairs to cytosine:guanine

The enzymes that transfer a methyl group to C5 of cytosine within specific sequences (C5 Mtases) deaminate the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) is omitted from the reaction. Recently, it was shown that cytosine deamination caused by C5 Mtases M.HpaII , M.SssI and M.MspI is enhanced in the presence of several analogues of SAM, and a mechanism for this analogue-promoted deamination was proposed. According to this mechanism, the analogues protonate C5 of the target cytosine, creating a dihydrocytosine intermediate that is susceptible to deamination. We show here that one of these analogues, 5 '-aminoadenosine (AA), enhances cytosine deamination by the Mtase M.EcoRII, but it does so without enhancing protonation of C5. Further, we show that uracil is an intermediate in the mutational pathway and propose an alternate mechanism for the analogue-promoted deamination. The new mechanism involves a facilitated water attack at C4 but does not require attack at C6 by the enzyme. The latter feature of the mechanism was tested by using M.EcoRII mutants defective in the nucleophilic attack at C6 in the deamination assay. We find that although these proteins are defective in methyl transfer and cytosine deamination, they cause cytosine deaminations in the presence of AA in the reaction. Our results point to a possible connection between the catalytic mechanism of C5 Mtases and of enzymes that transfer methyl groups to N4 of cytosine. Further, they provide an unusual example where a coenzyme activates an otherwise "dead" enzyme to perform catalysis by a new reaction pathway. These results were discussed in the first part of this dissertation.;Furthermore in the case of human, there are multiple uracil DNA glycosylases(UDGs) were reported. We would like to study the functions of different UDGs and their complementation of functions by cloning them into bacterial system. During this study, a special glycosylase activity in a methyl CpG-binding domain of the MBD family protein was discovered. The results were discussed in the second part of this dissertation.