PCNA clamp facilitates action of DNA cytosine methyltransferase 1 on hemimethylated DNA.
BACKGROUND
Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein known as a processivity factor of DNA polymerase delta. In addition to this role, PCNA interacts with a number of other proteins to increase their local concentration at replicated DNA sites. DNA cytosine methyltransferase 1 (Dnmt1), which preserves epigenetic signals by completing the methylation of hemimethylated DNA after DNA replication, has been indicated as one of these PCNA binding proteins by a previous work. However, the molecular mechanisms and functional significance of their association have not yet been studied.


RESULTS
Dnmt1 can be readily isolated from nuclear extracts by PCNA affinity chromatography. Studies of the interactions between the two proteins demonstrate that the N-terminal region of Dnmt1, which contains a typical PCNA binding motif, has core PCNA binding activity, and that the remaining portion of the protein exerts a negative influence on the interaction of Dnmt1 with PCNA. The affinity of Dnmt1 for DNA is much higher for DNA bound by PCNA than for free DNA. Furthermore, DNA methylation assays with hemimethylated DNA as a substrate revealed that PCNA clamp-bound DNA is methylated more efficiently by Dnmt1 than is free DNA.


CONCLUSIONS
These results provide the first biochemical evidence that physical interactions between PCNA and Dnmt1 facilitate the methylation of newly neplicated DNA, on which PCNA remains associated as a functional clamp.
D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation; D017931:DNA Primers; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D018411:Spodoptera; D020349:Surface Plasmon Resonance
D017931:DNA Primers; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse