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The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds

Abstract

The PWWP domain is a weakly conserved sequence motif found in >60 eukaryotic proteins, including the mammalian DNA methyltransferases Dnmt3a and Dnmt3b. These proteins often contain other chromatin-association domains. A 135-residue PWWP domain from mouse Dnmt3b (amino acids 223–357) has been structurally characterized at 1.8 Å resolution. The N-terminal half of this domain resembles a barrel-like five-stranded structure, whereas the C-terminal half contains a five-helix bundle. The two halves are packed against each other to form a single structural module that exhibits a prominent positive electrostatic potential. The PWWP domain alone binds DNA in vitro, probably through its basic surface. We also show that recombinant Dnmt3b2 protein (a splice variant of Dnmt3b) and two N-terminal deletion mutants (Δ218 and Δ369) have approximately equal methyl transfer activity on unmethylated and hemimethylated CpG-containing oligonucleotides. The Δ218 protein, which includes the PWWP domain, binds DNA more strongly than Δ369, which lacks the PWWP domain.

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Acknowledgements

We thank E. Li for providing mouse Dnmt3b2 cDNA, S. Pradhan for providing full-length Dnmt1; K.D. Wilkinson for help with analysis of DNA binding titration curves; P. Kearney for constructing overexpression plasmids; and L. Zhou, J.R. Horton, D. Schneider and R.M. Sweet for help with X-ray data collection. We also thank R.M. Blumenthal, T.H. Bestor and P.A. Wade for their critical comments on the manuscript. The study was supported in part by the National Institutes of Health grant to X.C.

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The authors declare no competing financial interests.

Correspondence to Xiaodong Cheng.

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Figure 1: Dnmt3 MTase family.
Figure 2: Structure of Dnmt3b2 PWWP domain.
Figure 3: DNA binding and methyl transfer activity of Dnmt3b2.
Figure 4: Comparison of PWWP domain with its structural homologs.
Figure 5: Views of defined elements in the Dnmt3b2 PWWP structure.
Figure 6: Sequence alignment of PWWP-containing proteins.