• Home
  • Search Results
  • DNA damage response factors from diverse pathways, including DNA crosslink repair, mediate alternative end joining.

DNA damage response factors from diverse pathways, including DNA crosslink repair, mediate alternative end joining.

PLoS genetics (2015-01-30)
Sean M Howard, Diana A Yanez, Jeremy M Stark
ABSTRACT

Alternative end joining (Alt-EJ) chromosomal break repair involves bypassing classical non-homologous end joining (c-NHEJ), and such repair causes mutations often with microhomology at the repair junction. Since the mediators of Alt-EJ are not well understood, we have sought to identify DNA damage response (DDR) factors important for this repair event. Using chromosomal break reporter assays, we surveyed an RNAi library targeting known DDR factors for siRNAs that cause a specific decrease in Alt-EJ, relative to an EJ event that is a composite of Alt-EJ and c-NHEJ (Distal-EJ between two tandem breaks). From this analysis, we identified several DDR factors that are specifically important for Alt-EJ relative to Distal-EJ. While these factors are from diverse pathways, we also found that most of them also promote homologous recombination (HR), including factors important for DNA crosslink repair, such as the Fanconi Anemia factor, FANCA. Since bypass of c-NHEJ is likely important for both Alt-EJ and HR, we disrupted the c-NHEJ factor Ku70 in Fanca-deficient mouse cells and found that Ku70 loss significantly diminishes the influence of Fanca on Alt-EJ. In contrast, an inhibitor of poly ADP-ribose polymerase (PARP) causes a decrease in Alt-EJ that is enhanced by Ku70 loss. Additionally, the helicase/nuclease DNA2 appears to have distinct effects from FANCA and PARP on both Alt-EJ, as well as end resection. Finally, we found that the proteasome inhibitor Bortezomib, a cancer therapeutic that has been shown to disrupt FANC signaling, causes a significant reduction in both Alt-EJ and HR, relative to Distal-EJ, as well as a substantial loss of end resection. We suggest that several distinct DDR functions are important for Alt-EJ, which include promoting bypass of c-NHEJ and end resection.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium chloride, BioPerformance Certified, ≥99% (titration), suitable for insect cell culture, suitable for plant cell culture
Supelco
DL-Dithiothreitol solution, 1 M in H2O
USP
Transplatin, United States Pharmacopeia (USP) Reference Standard
USP
Dehydrated Alcohol, United States Pharmacopeia (USP) Reference Standard
Supelco
Dehydrated Alcohol, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Sodium chloride, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Ethanol standards 10% (v/v), 10 % (v/v) in H2O, analytical standard
Sigma-Aldrich
Ethanol Fixative 80% v/v, suitable for fixing solution (blood films)
Sigma-Aldrich
Sodium azide, BioXtra
Sigma-Aldrich
Anti-Actin antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Ribonuclease A from bovine pancreas, (Solution of 50% glycerol, 10mM Tris-HCL pH 8.0)
Sigma-Aldrich
Sodium chloride solution, 5 M
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Propidium iodide, ≥94.0% (HPLC)
Sigma-Aldrich
Sodium azide, ReagentPlus®, ≥99.5%
Sigma-Aldrich
4′,6-Diamidino-2-phenylindole dihydrochloride, powder, BioReagent, suitable for cell culture, ≥98% (HPLC and TLC), suitable for fluorescence
Sigma-Aldrich
Sodium chloride, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
Sigma-Aldrich
Sodium chloride, tablet
Sigma-Aldrich
Sodium chloride, meets analytical specification of Ph. Eur., BP, USP, 99.0-100.5%
Sigma-Aldrich
Sodium chloride solution, 0.9% in water, BioXtra, suitable for cell culture
Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Sodium chloride solution, 0.85%
Sigma-Aldrich
Sodium azide, BioUltra, ≥99.5% (T)
Sigma-Aldrich
cis-Diamineplatinum(II) dichloride, ≥99.9% trace metals basis
Supelco
Ethanol solution, certified reference material, 2000 μg/mL in methanol
Sigma-Aldrich
Sodium chloride, random crystals, optical grade, 99.9% trace metals basis
Sigma-Aldrich
Sodium chloride-35Cl, 99 atom % 35Cl
Sigma-Aldrich
Sodium chloride, 99.999% trace metals basis
Sigma-Aldrich
Sodium chloride, AnhydroBeads, −10 mesh, 99.99% trace metals basis