Identification and characterization of interactions between de novo DNA methyltransferase 3b and thymine-DNA glycosylase(s)

The majority of methylation found in the vertebrate genome occurs at carbon 5 of the cytosine pyrimidine ring within CpG dinucleotides. DNA methylation patterns are established during early embryogenesis by the de novo methyltransferases, Dnmt3a and Dnmt3b. These patterns play a large role in genetic commitment and cellular differentiation via the heritable repression of certain developmental programs. An important in vivo function of Dnmt3b is methylation of the centromeric and pericentromeric satellite repeats, regions of heterochromatin that are rich in methylated CpG sites. Methylation of these repeats contributes to the higher-order heterochromatin structure that is essential to maintain genomic stability, repression of aberrant mitotic recombination and assurance of proper chromosome segregation. Mutations that ablate Dnmt3b activity, exemplified by those found in the autosomal recessive genetic disorder, ICF syndrome, lead to hypomethylation of the pericentromeric satellite repeats resulting in chromatin decondensation and enhanced chromosomal rearrangements.; A significant source of endogenous DNA damage is the spontaneous hydrolytic deamination of cytosine and 5-methylcytosine to uracil and thymidine, thereby generating U-G and T-G mismatches, respectively. If these promutagenic lesions are not repaired, C → T transition mutations occur during replication. Mechanisms to repair such damage have evolved in the form of mismatch-specific DNA glycosylases. G/T mismatch-specific thymine-DNA glycosylase (Tdg) initiates post-replicative base excision repair at T·G and U·G mismatches ultimately leading to reformation of the original C-G base pair.; Tdg was isolated as a positive protein interactor with Dnmt3b in a yeast two-hybrid screen. Subsequently, interaction between the endogenous proteins was demonstrated. It was determined that Dnmt3b and Tdg are targeted to genomic regions of heterochromatin. The regions/domains of Dnmt3b that mediate the interaction with Tdg were determined to be regions that have been previously identified as necessary for targeting Dnmt3b to pericentromeric heterochromatin. A T-G mismatch repair assay utilizing ES cells nullizygous for different DNA methyltransferases was developed to study the effect of DNA methyltransferases on base excision repair. Results of these experiments suggest that DNA methyltransferases potentiate T-G mismatch repair. During this course of investigation, it was determined that a putative RNA component is essential for proper T-G mismatch repair.