Functional Analysis of DNMT3A DNA Methyltransferase Mutations Reported in Patients with Acute Myeloid Leukemia

In mammals, DNA methylation is necessary for the maintenance of genomic stability, gene expression regulation, and other processes. During malignant diseases progression, changes in both DNA methylation patterns and DNA methyltransferase (MTase) genes are observed. Human de novo MTase DNMT3A is most frequently mutated in acute myeloid leukemia (AML) with a striking prevalence of R882H mutation, which has been extensively studied. Here, we investigate the functional role of the missense mutations (S714C, R635W, R736H, R771L, P777R, and F752V) found in the catalytic domain of DNMT3A in AML patients. These were accordingly mutated in the murine Dnmt3a catalytic domain (S124C, R45W, R146H, R181L, P187R, and F162V) and in addition, one-site CpG-containing DNA substrates were used as a model system. The 3-15-fold decrease (S124C and P187R) or complete loss (F162V, R45W, and R146H) of Dnmt3a-CD methylation activity was observed. Remarkably, Pro 187 and Arg 146 are not located at or near the Dnmt3a functional motives. Regulatory protein Dnmt3L did not enhance the methylation activity of R45W, R146H, P187R, and F162V mutants. The key steps of the Dnmt3a-mediated methylation mechanism, including DNA binding and transient covalent intermediate formation, were examined. There was a complete loss of DNA-binding affinity for R45W located in the AdoMet binding region and for R146H. Dnmt3a mutants studied in vitro suggest functional impairment of DNMT3A during pathogenesis

Impact of G-Quadruplex Structures on Methylation of Model Substrates by DNA Methyltransferase Dnmt3a

In mammals, de novo methylation of cytosines in DNA CpG sites is performed by DNA methyltransferase Dnmt3a. Changes in the methylation status of CpG islands are critical for gene regulation and for the progression of some cancers. Recently, the potential involvement of DNA G-quadruplexes (G4s) in methylation control has been found. Here, we provide evidence for a link between G4 formation and the function of murine DNA methyltransferase Dnmt3a and its individual domains. As DNA models, we used (i) an isolated G4 formed by oligonucleotide capable of folding into parallel quadruplex and (ii) the same G4 inserted into a double-stranded DNA bearing several CpG sites. Using electrophoretic mobility shift and fluorescence polarization assays, we showed that the Dnmt3a catalytic domain (Dnmt3a-CD), in contrast to regulatory PWWP domain, effectively binds the G4 structure formed in both DNA models. The G4-forming oligonucleotide displaced the DNA substrate from its complex with Dnmt3a-CD, resulting in a dramatic suppression of the enzyme activity. In addition, a direct impact of G4 inserted into the DNA duplex on the methylation of a specific CpG site was revealed. Possible mechanisms of G4-mediated epigenetic regulation may include Dnmt3a sequestration at G4 and/or disruption of Dnmt3a oligomerization on the DNA surface