Eukaryotic DNA methylation prevents genomic instability by regulating the expression of oncogenes and tumor-suppressor genes. The negative effects of dysregulated DNA methylation are highlighted by a strong correlation between mutations in the DNA methyltransferase gene α () and poor prognoses among acute myeloid leukemia (AML) patients. We show here that clinically observed mutations dramatically alter enzymatic activity, including mutations that lead to 6-fold hypermethylation and 3-fold hypomethylation of the human ( or ) gene promoter. Our results provide insights into the clinically observed heterogeneity of methylation in AML. Cytogenetically normal AML (CN-AML) constitutes 40-50% of all AML cases and is the most epigenetically diverse AML subtype with pronounced changes in non-CpG DNA methylation. We identified a subset of DNMT3A mutations that enhance the enzyme's ability to perform non-CpG methylation by 2-8-fold. Many of these mutations mapped to DNMT3A regions known to interact with proteins that themselves contribute to AML, such as thymine DNA glycosylase (TDG). Using functional mapping of TDG-DNMT3A interactions, we provide evidence that TDG and DNMT3-like (DNMT3L) bind distinct regions of DNMT3A. Furthermore, DNMT3A mutations caused diverse changes in the ability of TDG and DNMT3L to affect DNMT3A function. Cell-based studies of one of these DNMT3A mutations (S714C) replicated the enzymatic studies and revealed that it causes dramatic losses of genome-wide methylation. In summary, mutations in DNMT3A lead to diverse levels of activity, interactions with epigenetic machinery components and cellular changes.