Premium
An Investigation of Protein Function During DNA Repair in Bdelloid Rotifers
Author(s) -
McReynolds Jeanita Cherise,
Boerma Marjan,
Byrum Stephanie D,
Orr Lisa M,
Tackett Alan J,
Schurko Andrew
Publication year - 2017
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.31.1_supplement.906.8
Subject(s) - biology , rad51 , meiosis , dna repair , dna , microbiology and biotechnology , gene , genetics , proteome
Bdelloid rotifers are a class of aquatic microinvertebrates that have seemingly flourished for over 40 million years without sex, meiosis, or males. The long‐term success of this ancient ameiotic lineage is attributed, in part, to the extraordinary ability of bdelloids to repair DNA double strand breaks induced by desiccation. Our objective is to characterize proteins and their interactions during DNA repair in bdelloid rotifers. We first targeted meiotic proteins as a group of candidate DNA repair proteins. Despite the longstanding absence of meiosis in bdelloids, their genomes contain four genes that code for proteins (SPO11, HOP1, MSH4, MSH5) specific to meiosis in other eukaryotes. Western blot assays using custom antibodies for SPO11, HOP1, MSH4 and MSH5 were done to confirm that their corresponding genes were expressing proteins in the bdelloid Adineta vaga . To characterize binding partners of these meiotic proteins, protein pull‐down assays were then done. We generated 6×histidine‐tagged copies of each meiotic protein (and also of DNA repair proteins RAD51 and MRE11) and these were used in pull‐down assays with Dynabeads and protein lysates from A. vaga . SDS‐PAGE and mass spectrometry were used to identify potential binding partners. Next, to further our investigation into DNA repair proteins, we also identified other candidate DNA repair proteins using a whole proteome analysis of irradiated and non‐irradiated cultures of A. vaga with high‐resolution mass spectrometry. To induce DNA double‐strand breaks, bdelloid cultures were irradiated with a total dose of 280 Gray of ionizing radiation using a cesium‐137 source. Proteins were then isolated from these bdelloids and identified using tandem mass tag labeling and high‐resolution mass spectrometric analysis with a Thermo Velos Orbitrap mass spectrometer. These proteins will the targeted for future analyses using pull‐downs, yeast two‐hybrid and other techniques to further examine their role in DNA repair. Overall, this study aims to identify candidate proteins and characterize their roles in DNA repair in bdelloids, which will further our understanding of this process in rotifers and other eukaryotes. Support or Funding Information This work was supported by grants from the National Center for Research Resources (5P20RR016460) & National Institute of General Medical Sciences (8P20GM103429) from the NIH.