ICF syndrome mutations cause a broad spectrum of biochemical defects in DNMT3B-mediated de novo DNA methylation. 
The DNMT3B de novo DNA methyltransferase (DNMT) plays a major role in establishing DNA methylation patterns in early mammalian development, but its catalytic mechanism remains poorly characterized.  Here, we provide a comprehensive biochemical analysis of human DNMT3B function through the characterization of a series of site-directed DNMT3B variants associated with immunodeficiency, centromere instability, and facial anomalies (ICF) syndrome.  Our data reveal several novel and important aspects of DNMT3B function.  First, DNMT3B, unlike DNMT3A, requires a DNA cofactor in order to stably bind to S-adenosyl-l-methionine (SAM), suggesting that it proceeds according to an ordered catalytic scheme.  Second, ICF mutations cause a broad spectrum of biochemical defects in DNMT3B function, including defects in homo-oligomerization, SAM binding, SAM utilization, and DNA binding.  Third, all tested ICF mutations, including the A766P and R840Q variants, result in altered catalytic properties without interfering with DNMT3L-mediated stimulation; this indicates that DNMT3L is not involved in the pathogenesis of ICF syndrome.  Finally, our study reveals a novel level of coupling between substrate binding, oligomerization, and catalysis that is likely conserved within the DNMT3 family of enzymes.