Abstract
DNA methylation patterns are the dynamic outcome of antagonist methylation and demethylation mechanisms, but the latter are still poorly understood. Active DNA demethylation in plants is mediated by a family of DNA glycosylases typified by Arabidopsis ROS1 (repressor of silencing 1). ROS1 and its homologs remove 5-methylcytosine and incise the sugar backbone at the abasic site, thus initiating a base excision repair pathway that finally inserts an unmethylated cytosine. The DNA 3'-phosphatase ZDP processes some of the incision products generated by ROS1, allowing subsequent DNA polymerization and ligation steps. In this work, we examined the possible role of plant XRCC1 (x-ray cross-complementing group protein 1) in DNA demethylation. We found that XRCC1 interacts in vitro with ROS1 and ZDP and stimulates the enzymatic activity of both proteins. Furthermore, extracts from xrcc1 mutant plants exhibit a reduced capacity to complete DNA demethylation initiated by ROS1. An anti-XRCC1 antibody inhibits removal of the blocking 3'-phosphate in the single-nucleotide gap generated during demethylation and reduces the capacity of Arabidopsis cell extracts to ligate a nicked DNA intermediate. Our results suggest that XRCC1 is a component of plant base excision repair and functions at several stages during active DNA demethylation in Arabidopsis.
Highlights
Active DNA demethylation in plants is initiated by 5-meC DNA glycosylases of the ROS1/DME family and continued through a base excision repair pathway
We confirmed that His-ROS1 was bound by maltose-binding protein (MBP)-XRCC1 immobilized on an amylose column (Fig. 1B)
To define ROS1 regions implicated in the interaction, we generated a series of N- and C-terminal His-ROS1 deletion mutants, and the purified proteins were bound to a nickel-Sepharose resin and used to analyze their capacity to bind MBP-XRCC1 (Fig. 1, C and D)
Summary
Active DNA demethylation in plants is initiated by 5-meC DNA glycosylases of the ROS1/DME family and continued through a base excision repair pathway. Active DNA demethylation in plants is mediated by a family of DNA glycosylases typified by Arabidopsis ROS1 (repressor of silencing 1). An anti-XRCC1 antibody inhibits removal of the blocking 3-phosphate in the single-nucleotide gap generated during demethylation and reduces the capacity of Arabidopsis cell extracts to ligate a nicked DNA intermediate. Our results suggest that XRCC1 is a component of plant base excision repair and functions at several stages during active DNA demethylation in Arabidopsis. The DNA 3Ј-phosphatase ZDP functions downstream of ROS1 by removing the blocking 3Ј-phosphate present in ,␦-elimination products and allowing subsequent DNA polymerization and ligation steps [16]. Our results suggest that XRCC1 functions during active DNA demethylation in Arabidopsis
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