Identification of base excision repair protein complexes

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Robert W. Sobol  

PROVIDER: MSV000094615 | MassIVE | Wed Apr 24 20:18:00 BST 2024

SECONDARY ACCESSION(S): PXD051729

REPOSITORIES: MassIVE