Figure 3.

The P705S mutation severely impairs the ability of Dnmt3a to methylate DNA

(a) Sequence alignment of a portion of human and mouse Dnmt3a. P709 in human Dnmt3a corresponds to P705 in mouse Dnmt3a.(b) Myc-tagged mouse Dnmt3a2 or Dnmt3a2:P705S (PS) mutant was transfected into late-passage Dnmt3a/3b double knockout (7aabb) ES cells, and stable clones obtained. The cell lysates of these clones, as well as untransfected 7aabb cells, were analysed by immunoblotting with anti-Myc and anti-β-actin antibodies. The dotted line indicates where the membrane was cut.(c) Genomic DNA from cells described in (b), as well as WT (J1) ES cells, was digested with MaeII and analysed by Southern hybridization with a probe specific for the major satellite repeats (MSRs). Densitometry was used to determine the relative methylation levels (methylation scores).