Genetic and Biochemical Characterization of Drosophila Gen during DNA Repair and Recombination Public Deposited

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  • March 19, 2019
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  • Bellendir, Stephanie
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
Abstract
  • Holliday junction (HJ) resolvases maintain genome stability by processing DNA intermediates that arise during DNA repair. While human GEN1 and several orthologs possess HJ resolvase activity in vitro, in vivo studies indicate that GEN1 (and S. cerevisiae Yen1) is secondary to the Mus81–Mms4/Eme1 nuclease. Prior work suggests that this relationship is reversed in Drosophila; however, a full characterization of Gen has yet to be performed. Here we confirm that Gen is the primary HJ resolvase in Drosophila somatic cells and reveal key elements of its biochemistry, including that it preferentially cuts 5’ flaps and exists in a monomer-dimer equilibrium. We found that Gen mutants are hypersensitive to a variety of DNA damaging agents relative to mus81 mutants. However, like the human and yeast orthologs, Gen is primarily or exclusively cytoplasmic during interphase. We next purified recombinant Gen and observed robust activity on fixed, mobile, and nicked HJs, as well as on 5’ flaps and replication fork-like structures. Our kinetic studies of Gen with 5’ flaps and HJs indicate that unlike its orthologs, Gen cuts the 5’ flap structure faster than the HJ structure, even at vast excess protein. Unexpectedly, our kinetic data suggests that dimerization accelerates cleavage of the flap. We verified dimerization by atomic force microscopy and gel-shift assays and show that Gen exists in a monomer-dimer equilibrium. In conclusion, we suggest that although HJs represent an important substrate for Gen, flaps and replication fork derivatives may be more relevant than HJs when considering the in vivo relationship between Gen and MUS81.
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  • In Copyright
Advisor
  • Ramsden, Dale
  • Copenhaver, Gregory
  • Erie, Dorothy
  • Jones, Corbin
  • Sekelsky, Jeff
  • Ahmed, Shawn
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2015
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  • Chapel Hill, NC
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