Affiliation:
1. Department of Urology and Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, California 90033
Abstract
ABSTRACT
It has been hypothesized that protein factors may protect CpG islands from methyltransferase during development and that demethylation may involve protein-DNA interactions at demethylated sites. However, direct evidence has been lacking. In this study, demethylation at the EBNA-1 binding sites of the Epstein-Barr virus latent replication origin,
oriP
, was investigated by using human cells. Several novel findings are discussed. First, there are specific preferential demethylation sites within the
oriP
region. Second, the DNA sequence of
oriP
alone is not the target of an active demethylation process. Third, EBNA-1 binding is required for the site-specific demethylation in
oriP
. Interestingly, CpG sites adjacent to and between the EBNA-1 sites do not become demethylated. Fourth, demethylation of the first DNA strand in
oriP
at the EBNA-1 binding sites involves a passive (replication-dependent) mechanism. The second-strand demethylation appears to occur through an active mechanism. That is, EBNA-1 protein binding prevents the EBNA-1 binding sites from being remethylated after one round of DNA replication, and it appears that an active demethylase then demethylates these hemimethylated sites. This study provides clear evidence that protein binding specifies sites of DNA demethylation and provides insights into the sequence of steps and the mechanism of demethylation.
Publisher
American Society for Microbiology
Subject
Cell Biology,Molecular Biology
Cited by
83 articles.
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