APN2 6C101 !!EXCLUSIVE!!
Ribonucleoside monophosphates (rNMPs) mis-incorporated during DNA replication are removed by RNase H2-dependent excision repair or by topoisomerase I (Top1)-catalyzed cleavage. The cleavage of rNMPs by Top1 produces 3′ ends harboring terminal adducts, such as 2′,3′-cyclic phosphate or Top1 cleavage complex (Top1cc), and leads to frequent mutagenesis and DNA damage checkpoint induction. We surveyed a range of candidate enzymes from Saccharomyces cerevisiae for potential roles in Top1-dependent genomic rNMP removal. Genetic and biochemical analyses reveal that Apn2 resolves phosphotyrosine-DNA conjugates, terminal 2′,3′-cyclic phosphates, and their hydrolyzed products. APN2 also suppresses 2-base pair (bp) slippage mutagenesis in RNH201-deficient cells. Our results define additional activities of Apn2 in resolving a wide range of 3′ end blocks and identify a role for Apn2 in maintaining genome integrity during rNMP repair.
An AP endonuclease belongs to the Exo III family of enzymes. The function of AP endonucleases is to remove DNA 3′-terminal phosphate groups from an internally located AP lesion via a wedge-shaped cleavage that is expected to generate a 5′-phosphate at the 3′ side of the original lesion. We report the first crystal structure of yeast AP endonuclease Apn2 in complex with DNA and propose a mechanism for Apn2-mediated cleavage. The structure reveals that Apn2 adopts an alphabeta/EF-hand protein fold similar to that of the other members of the family, and binds to DNA via a canonical “head-to-tail” dimer, in which the two motifs interact through extensive contacts. We propose that the wedge cleavage mechanism is enabled by a conformation change at the interaction surface between subunits and the major groove of DNA, which results in repositioning of the conserved residues in the respective motifs. This makes possible binding to the DNA and subsequent scissile phosphodiester bond cleavage. Potential homologues of Apn2 are found throughout the phylum of Eukarya, and we show that mouse AP endonuclease is involved in cell-fate determination during development.
Leave a Reply