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Citation
Bellendir, S.P., Rognstad, D.J., Morris, L.P., Zapotoczny, G., Walton, W.G., Redinbo, M.R., Ramsden, D.A., Sekelsky, J., Erie, D.A. (2017). Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates.  Nucleic Acids Res. 45(9): 5333--5348.
FlyBase ID
FBrf0235593
Publication Type
Research paper
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.
PubMed ID
PubMed Central ID
PMC5435919 (PMC) (EuropePMC)
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Secondary IDs
    Language of Publication
    English
    Additional Languages of Abstract
    Parent Publication
    Publication Type
    Journal
    Abbreviation
    Nucleic Acids Res.
    Title
    Nucleic Acids Research
    Publication Year
    1974-
    ISBN/ISSN
    0305-1048
    Data From Reference
    Aberrations (1)
    Alleles (2)
    Genes (2)
    Physical Interactions (2)
    Cell Lines (1)