Premium
Cell Cycle‐Dependent Cleaving of Holliday Junctions via Yen1 in S. cerevisiae
Author(s) -
Hadi Hafridha,
Fischhaber Paula
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.08929
Subject(s) - biology , ura3 , holliday junction , plasmid , population , dna , genetics , saccharomyces cerevisiae , microbiology and biotechnology , homologous recombination , gene , demography , sociology
Double‐strand breaks (DSBs) in DNA can be harmful if left unrepaired. Eukaryotes use repair mechanisms, some of which involve intact chromosomes that template repair via an intertwined intermediate known as a Holliday Junction (HJ). In S. cerevisiae , recruitment of the endonuclease enzymes that cleave HJs back into individual chromosomes is not well understood. Yen1 resolves HJs but it’s unclear whether it localizes to anaphase bridges, the final intertwined regions during chromosomal separation. Yen1 appears in anaphase which occurs during a small time frame within the cell cycle, making it difficult to observe in an asynchronous cell population. Further, Yen1 is present in lower abundance relative to other proteins, therefore, microscopy experiments with fluorescently labeled Yen1 will suffer from low signal‐to‐noise. Our aim is to clone a yeast strain with a bright version of red fluorescent protein under the strong galactose ( GAL1 ) promoter so that Yen1‐yEMRFP will be in higher abundance, thereby allowing us to track the position and timing Yen1 during HJ resolution. To construct this strain, we used adaptamer‐mediated Polymerase Chain Reaction (PCR), DNA digestions and ligations to prepare two DNA plasmids. Plasmid One contains a gene for yeast Enhanced Monomeric Red Fluorescent Protein (yEMRFP), a partial URA3 auxotrophic selection marker gene, and an upstream flanker sequence of the target yeast chromosome to direct integration. Plasmid Two contains the other half of the partial URA3 selection marker, the galactose promoter, and the downstream flanker sequence to target integration. Candidate clones were screened by PCR and DNA sequencing which showed that the candidate plasmids had the desired sequences and are ready to be integrated into the yeast chromosome. These DNA plasmids will allow cloning of a specialized yeast strain that will produce an improved signal‐to‐noise resolution to elucidate Yen1’s role at anaphase bridges. Support or Funding Information This research was funded by NIH grant, SC1GM127204.