title: Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates.
abstract: Human GEN1 and yeast Yen1 are endonucleases with the ability to cleave Holliday junctions (HJs), which are proposed intermediates in recombination. In vivo, GEN1 and Yen1 function secondarily to Mus81, which has weak activity on intact HJs. We show that the genetic relationship is reversed in Drosophila, with Gen mutants having more severe defects than mus81 mutants. In vitro, DmGen, like HsGEN1, efficiently cleaves HJs, 5΄ flaps, splayed arms, and replication fork structures. We find that the cleavage rates for 5΄ flaps are significantly higher than those for HJs for both DmGen and HsGEN1, even in vast excess of enzyme over substrate. Kinetic studies suggest that the difference in cleavage rates results from a slow, rate-limiting conformational change prior to HJ cleavage: formation of a productive dimer on the HJ. Despite the stark difference in vivo that Drosophila uses Gen over Mus81 and humans use MUS81 over GEN1, we find the in vitro activities of DmGen and HsGEN1 to be strikingly similar. These findings suggest that simpler branched structures may be more important substrates for Gen orthologs in vivo, and highlight the utility of using the Drosophila model system to further understand these enzymes.
authors.affiliation: Curriculum in Genetics and Molecular Biology, Chapel Hill, NC 27599, USA.
authors.forename: Stephanie P
authors.initials: SP
authors.lastname: Bellendir
authors.affiliation: Curriculum in Genetics and Molecular Biology, Chapel Hill, NC 27599, USA.
authors.forename: Danielle J
authors.initials: DJ
authors.lastname: Rognstad
authors.affiliation: Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27599, USA.
authors.forename: Lydia P
authors.initials: LP
authors.lastname: Morris
authors.affiliation: Curriculum in Genetics and Molecular Biology, Chapel Hill, NC 27599, USA.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Zapotoczny
authors.affiliation: Department of Chemistry, Chapel Hill, NC 27599, USA.
authors.forename: William G
authors.initials: WG
authors.lastname: Walton
authors.affiliation: Department of Chemistry, Chapel Hill, NC 27599, USA.|Integrative Program for Biological and Genome Sciences, Chapel Hill, NC 27599, USA.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Redinbo
authors.affiliation: Curriculum in Genetics and Molecular Biology, Chapel Hill, NC 27599, USA.|Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27599, USA.|Department of Biochemistry and Biophysics, Chapel Hill, NC 27599, USA.
authors.forename: Dale A
authors.initials: DA
authors.lastname: Ramsden
authors.affiliation: Curriculum in Genetics and Molecular Biology, Chapel Hill, NC 27599, USA.|Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27599, USA.|Integrative Program for Biological and Genome Sciences, Chapel Hill, NC 27599, USA.|Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Jeff
authors.initials: J
authors.lastname: Sekelsky
authors.affiliation: Department of Chemistry, Chapel Hill, NC 27599, USA.|Integrative Program for Biological and Genome Sciences, Chapel Hill, NC 27599, USA.
authors.forename: Dorothy A
authors.initials: DA
authors.lastname: Erie
mesh_terms: D000818:Animals; D001483:Base Sequence; D003593:Cytoplasm / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D045566:DNA, Cruciform / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism*; D045565:Holliday Junction Resolvases / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D055503:Protein Multimerization; D021381:Protein Transport; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C495865:MUS81 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D004720:Endonucleases; C494357:GEN protein, Drosophila; C533748:GEN1 protein, human; D045565:Holliday Junction Resolvases