Holliday junction (HJ) resolution is essential for chromosome segregation at meiosis and the repair of stalled/collapsed replication forks in mitotic cells. All organisms possess nucleases that promote HJ resolution by the introduction of symmetrically related nicks in two strands at, or close to, the junction point. GEN1, a member of the Rad2/XPG nuclease family, was isolated recently from human cells and shown to promote HJ resolution in vitro and in vivo. Here, we provide the first biochemical/structural characterization of GEN1, showing that, like the Escherichia coli HJ resolvase RuvC, it binds specifically to HJs and resolves them by a dual incision mechanism in which nicks are introduced in the pair of continuous (noncrossing) strands within the lifetime of the GEN1–HJ complex. In contrast to RuvC, but like other Rad2/XPG family members such as FEN1, GEN1 is a monomeric 5′-flap endonuclease. However, the unique feature of GEN1 that distinguishes it from other Rad2/XPG nucleases is its ability to dimerize on HJs. This functional adaptation provides the two symmetrically aligned active sites required for HJ resolution.
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