The enzymatic properties of Arabidopsis thaliana DNA polymerase λ suggest a role in base excision repair.

Base excision repair (BER) generates gapped DNA intermediates containing a 5'-terminal 2-deoxyribose-5-phosphate (5'-dRP) group. In mammalian cells, gap filling and dRP removal are catalyzed by Pol ß, which belongs to the X family of DNA polymerases. In higher plants, the only member of the X family of DNA polymerases is Pol λ. Although it is generally believed that plant Pol λ participates in BER, there is limited experimental evidence for this hypothesis. Here we have characterized the biochemical properties of Arabidopsis thaliana Pol λ (AtPol λ) in a BER context, using a variety of DNA repair intermediates. We have found that AtPol λ performs gap filling inserting the correct nucleotide, and that the rate of nucleotide incorporation is higher in substrates containing a C in the template strand. Gap filling catalyzed by AtPol λ is most efficient with a phosphate at the 5'-end of the gap and is not inhibited by the presence of a 5'-dRP mimic. We also show that AtPol λ possesses an intrinsic dRP lyase activity that is reduced by mutations at two lysine residues in its 8-kDa domain, one of which is present in Pol λ exclusively and not in any Pol ß homolog. Importantly, we also found that the dRP lyase activity of AtPol λ allows efficient completion of uracil repair in a reconstituted short-patch BER reaction. These results suggest that AtPol λ plays an important role in plant BER.

The C-terminal domain of Arabidopsis ROS1 DNA demethylase interacts with histone H3 and is required for DNA binding and catalytic activity.

Active DNA demethylation plays an important role in controlling methylation patterns in eukaryotes. In plants, the DEMETER-LIKE (DML) family of 5-methylcytosine DNA glycosylases initiates DNA demethylation through a base excision repair pathway. However, it is poorly understood how these DNA demethylases are recruited to their target loci and the role that histone marks play in this process. Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) is a representative enzyme of the DML family, whose members are uniquely characterized by a basic amino-terminal domain mediating nonspecific binding to DNA, a discontinuous catalytic domain, and a conserved carboxy-terminal domain of unknown function. Here, we show that ROS1 interacts with the N-terminal tail of H3 through its C-terminal domain. Importantly, phosphorylation at H3 Ser28, but not Ser10, abrogates ROS1 interaction with H3. Conserved residues at the C-terminal domain are not only required for H3 interaction, but also for efficient DNA binding and catalytic activity. Our findings suggest that the C-terminal domain of ROS1 may function as a histone reader module involved in recruitment of the DNA demethylase activity to specific genomic regions.