10779/crick.12652109.v1 Ying Wai Chan Ying Wai Chan Stephen West Stephen West GEN1 promotes Holliday junction resolution by a coordinated nick and counter-nick mechanism The Francis Crick Institute 2020 DNA Cleavage DNA, Cruciform Holliday Junction Resolvases Humans Protein Multimerization Substrate Specificity West Developmental Biology 05 Environmental Sciences 06 Biological Sciences 08 Information and Computing Sciences 2020-07-14 12:07:58 Journal contribution https://crick.figshare.com/articles/journal_contribution/GEN1_promotes_Holliday_junction_resolution_by_a_coordinated_nick_and_counter-nick_mechanism/12652109 Holliday junctions (HJs) that physically link sister chromatids or homologous chromosomes are formed as intermediates during DNA repair by homologous recombination. Persistent recombination intermediates are acted upon by structure-selective endonucleases that are required for proper chromosome segregation at mitosis. Here, we have purified full-length human GEN1 protein and show that it promotes Holliday junction resolution by a mechanism that is analogous to that exhibited by the prototypic HJ resolvase E. coli RuvC. We find that GEN1 cleaves HJs by a nick and counter-nick mechanism involving dual co-ordinated incisions that lead to the formation of ligatable nicked duplex products. As observed with RuvC, cleavage of the first strand is rate limiting, while second strand cleavage is rapid. In contrast to RuvC, however, GEN1 is largely monomeric in solution, but dimerizes on the HJ. Using HJs containing non-cleavable phosphorothioate-containing linkages in one strand, we show that the two incisions can be uncoupled and that the first nick occurs upon GEN1 dimerization at the junction. These results indicate that the mechanism of HJ resolution is largely conserved from bacteria to man, despite a lack of sequence homology between the resolvases.