MassIVE MSV000094615

Partial Public PXD051729

Identification of base excision repair protein complexes

Description

We recapitulated two main, cellular base excision repair (BER) complexes (herein termed Complex A, comprised of POLB and XRCC1 and Complex B, XRCC1-devoid complexes) by exploiting a separation-of-function mutant of POLB that does not bind to XRCC1, the V303-loop mutant POLB(TM). Cell lines were developed that expressed Flag-tagged-POLB(WT) and Flag-tagged-POLB(TM). To screen for binding partners of POLB or the POLB/XRCC1 complex, anti-Flag M2 affinity gel was used to immunoprecipitate proteins from cell lysates of LN428/Flag-POLB(WT), LN428/Flag-POLB(TM) or LN428/EGFP cells. The immunoprecipitates were loaded on SDS-PAGE gels and proteins were separated from low molecular weight reagents using short gel (~1cm) fractionation. The gels were stained by Coomassie blue. Gel regions were excised as indicated and processed for tryptic digestion. Liquid-chromatography Fourier transform mass spectrometry was used to measure the mass-to-charge (m/z) ratio, retention time, and intensity of more than 100,000 peptide signals that are detected in high-resolution full-scan mass spectra. [doi:10.25345/C5Z02ZM13] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: TRIP12 ; POLB ; Base Excision Repair ; DNA Damage Response ; Repair pathway choice

Contact

Principal Investigators:
(in alphabetical order)
Robert W. Sobol, Brown University, USA
Submitting User: xuzst11
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