Mutations in the Arabidopsis ROS1 locus cause transcriptional silencing of a transgene and a homologous endogenous gene. In the ros1 mutants, the promoter of the silenced loci is hypermethylated, which may be triggered by small RNAs produced from the transgene repeats. The transcriptional silencing in ros1 mutants can be released by the ddm1 mutation or the application of the DNA methylation inhibitor 5-aza-2'-deoxycytidine. ROS1 encodes an endonuclease III domain nuclear protein with bifunctional DNA glycosylase/lyase activity against methylated but not unmethylated DNA. The ros1 mutant shows enhanced sensitivity to genotoxic agents methyl methanesulfonate and hydrogen peroxide. We suggest that ROS1 is a DNA repair protein that represses homology-dependent transcriptional gene silencing by demethylating the target promoter DNA.

Cytosine methylation is an epigenetic mark that promotes gene silencing and plays important roles in development and genome defense against transposons. Methylation patterns are established and maintained by DNA methyltransferases that catalyze transfer of a methyl group from S-adenosyl-L-methionine to cytosine bases in DNA. Erasure of cytosine methylation occurs during development, but the enzymatic basis of active demethylation remains controversial. In Arabidopsis thaliana, DEMETER (DME) activates the maternal expression of two imprinted genes silenced by methylation, and REPRESSOR OF SILENCING 1 (ROS1) is required for release of transcriptional silencing of a hypermethylated transgene. DME and ROS1 encode two closely related DNA glycosylase domain proteins, but it is unknown whether they participate directly in a DNA demethylation process or counteract silencing through an indirect effect on chromatin structure. Here we show that DME and ROS1 catalyze the release of 5-methylcytosine (5-meC) from DNA by a glycosylase͞lyase mechanism. Both enzymes also remove thymine, but not uracil, mismatched to guanine. DME and ROS1 show a preference for 5-meC over thymine in the symmetric dinucleotide CpG context, where most plant DNA methylation occurs. Nevertheless, they also have significant activity on both substrates at CpApG and asymmetric sequences, which are additional methylation targets in plant genomes. These findings suggest that a function of ROS1 and DME is to initiate erasure of 5-meC through a base excision repair process and provide strong biochemical evidence for the existence of an active DNA demethylation pathway in plants.Arabidopsis ͉ demethylation ͉ gene silencing ͉ methylation M ethylation of cytosine at carbon 5 of the pyrimidine ring [5-methylcytosine (5-meC)] is an epigenetic modification that guides formation of transcriptionally silent chromatin and allows transmission of specific patterns of gene activity across cellular divisions (1, 2). In eukaryotes, DNA methylation is detected in protists, fungi, plants, and animals (3) and plays important roles in the establishment of developmental programs (4, 5) and in genome defense against parasitic mobile elements (6). Most of mammalian and plant DNA methylation is restricted to symmetrical CpG sequences, but plants also have significant levels of cytosine methylation in the symmetric context CpNpG (where N is any nucleotide) and even in asymmetric contexts (2, 7). Similarly to other biochemical modifications such as protein phosphorylation and acetylation, DNA methylation is also reversible. Demethylation may take place as a passive process because of lack of maintenance methylation during several cycles of DNA replication or as an active mechanism in the absence of replication (8). In mammalian preimplantation embryos the maternal genome is demethylated by a passive process along cleavage stages, whereas the paternal genome is demethylated by an active mechanism immediately after fertilization (9). In addition to global demethylation, site-specific...

Cytosine DNA methylation is a stable epigenetic mark for maintenance of gene silencing across cellular divisions, but it is a reversible modification. Genetic and biochemical studies have revealed that the Arabidopsis DNA glycosylase domain-containing proteins ROS1 (REPRESSOR OF SILENCING 1) and DME (DEMETER) initiate erasure of 5-methylcytosine through a base excision repair process. The Arabidopsis genome encodes two paralogs of ROS1 and DME, referred to as DEMETER-LIKE proteins DML2 and DML3. We have found that DML2 and DML3 are 5-methylcytosine DNA glycosylases that are expressed in a wide range of plant organs. We analyzed the distribution of methylation marks at two methylated loci in wild-type and dml mutant plants. Mutations in DML2 and/or DML3 lead to hypermethylation of cytosine residues that are unmethylated or weakly methylated in wild-type plants. In contrast, sites that are heavily methylated in wild-type plants are hypomethylated in mutants. These results suggest that DML2 and DML3 are required not only for removing DNA methylation marks from improperly-methylated cytosines, but also for maintenance of high methylation levels in properly targeted sites.

SUMMARY DNA methylation is an important epigenetic mark established by the combined actions of methylation and demethylation reactions. Plants use a base excision repair pathway for active DNA demethylation. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase ROS1 incises the DNA backbone and part of the product has a single-nucleotide gap flanked by 3′- and 5′-phosphate termini. Here we show that the DNA phosphatase ZDP removes the blocking 3′-phosphate, allowing subsequent DNA polymerization and ligation steps needed to complete the repair reactions. ZDP and ROS1 interact in vitro and co-localize in vivo in nucleoplasmic foci. Extracts from zdp mutant plants are unable to complete DNA demethylation in vitro, and the mutations cause DNA hypermethylation and transcriptional silencing of a reporter gene. Genome-wide methylation analysis in zdp mutant plants identified hundreds of hypermethylated endogenous loci. Our results show that ZDP functions downstream of ROS1 in one branch of the active DNA demethylation pathway.

Base excision repair (BER) is a critical pathway in cellular defense against endogenous or exogenous DNA damage. This elaborate multistep process is initiated by DNA glycosylases that excise the damaged base, and continues through the concerted action of additional proteins that finally restore DNA to the unmodified state. BER has been subject to detailed biochemical analysis in bacteria, yeast and animals, mainly through in vitro reproduction of the entire repair reaction in cell-free extracts. However, an understanding of this repair pathway in plants has consistently lagged behind. We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro. We have used this system to demonstrate that Arabidopsis cell extracts contain the enzymatic machinery required to completely repair ubiquitous DNA lesions, such as uracil and abasic (AP) sites. Our results reveal that AP sites generated after uracil excision are processed both by AP endonucleases and AP lyases, generating either 5′- or 3′-blocked ends, respectively. We have also found that gap filling and ligation may proceed either through insertion of just one nucleotide (short-patch BER) or several nucleotides (long-patch BER). This experimental system should prove useful in the biochemical and genetic dissection of BER in plants, and contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways.