Figure 2.

Mutation of the PBD abolishes Dnmt1 interaction with PCNA and prevents accumulation at replication sites in early and mid S-phase. (A, B) Confocal mid sections of living mouse C2C12 myoblasts expressing either GFP-Dnmt1^wt (A) or GFP-Dnmt1^Q162E (B). Cells were co-transfected with RFP-PCNA to identify replication foci and to distinguish S-phase stages. Scale bars: 5 µm. GFP-Dnmt1^wt accumulates at replication sites throughout S phase where it co-localizes with RFP-PCNA. In G2 cells a fraction of Dnmt1 remains associated with the late replicating pericentric heterochromatin. In contrast, GFP-Dnmt1^Q162E shows a fully dispersed nuclear distribution in early and mid S-phase stages, whereas in late S-phase and G2 association with centromeric heterochromatin is still observed. (C) Endogenous PCNA efficiently co-immunoprecipitates with GFP-Dnmt1^wt but not with GFP-Dnmt1^Q162E. Cell extracts were prepared from HEK 293T cells expressing either GFP-Dnmt1^wt or GFP-Dnmt1^Q162E. Precipitated proteins were detected by immunostaining with antibodies against GFP and PCNA, respectively.