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Molecular and Cellular Biology, February 2002, p. 704-723, Vol. 22, No. 3
0270-7306/01/$04.00+0     DOI: 10.1128/MCB.22.3.704-723.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Murine De Novo Methyltransferase Dnmt3a Demonstrates Strand Asymmetry and Site Preference in the Methylation of DNA In Vitro

Iping G. Lin, Li Han, Alexander Taghva, Laura E. O’Brien, and Chih-Lin Hsieh*

Department of Urology and Department of Biochemistry and Molecular Biology, University of Southern California, Norris Cancer Center, Los Angeles, California 90033

Received 27 July 2001/ Returned for modification 27 September 2001/ Accepted 23 October 2001

CpG methylation is involved in a wide range of biological processes in vertebrates as well as in plants and fungi. To date, three enzymes, Dnmt1, Dnmt3a, and Dnmt3b, are known to have DNA methyltransferase activity in mouse and human. It has been proposed that de novo methylation observed in early embryos is predominantly carried out by the Dnmt3a and Dnmt3b methyltransferases, while Dntm1 is believed to be responsible for maintaining the established methylation patterns upon replication. Analysis of the sites methylated in vivo using the bisulfite genomic sequencing method confirms the previous finding that some regions of the plasmid are much more methylated by Dnmt3a than other regions on the same plasmid. However, the preferred targets of the enzyme cannot be determined due to the presence of other methylases, DNA binding proteins, and chromatin structure. To discern the DNA targets of Dnmt3a without these compounding factors, sites methylated by Dnmt3a in vitro were analyzed. These analyses revealed that the two cDNA strands have distinctly different methylation patterns. Dnmt3a prefers CpG sites on a strand in which it is flanked by pyrimidines over CpG sites flanked by purines in vitro. These findings indicate that, unlike Dnmt1, Dnmt3a most likely methylates one strand of DNA without concurrent methylation of the CpG site on the complementary strand. These findings also indicate that Dnmt3a may methylate some CpG sites more frequently than others, depending on the sequence context. Methylation of each DNA strand independently and with possible sequence preference is a novel feature among the known DNA methyltransferases.

* Corresponding author. Mailing address: Department of Urology and Department of Biochemistry and Molecular Biology, University of Southern California, 1441 Eastlake Ave., Room 5420, Norris Cancer Center, Los Angeles, CA 90033. Phone: (323) 865-0567. Fax: (323) 865-3019. E-mail: hsieh_c{at}ccnt.hsc.usc.edu.

Molecular and Cellular Biology, February 2002, p. 704-723, Vol. 22, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/MCB.22.3.704-723.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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