Proteomic approach to identification of proteins reactive for abasic sites in DNA.
Apurinic/apyrimidinic (AP) sites, a prominent type of DNA damage, are repaired through the base excision repair mechanism in both prokaryotes and eukaryotes and may interfere with many other cellular processes. A full repertoire of AP site-binding proteins in cells is presently unknown, preventing reliable assessment of harm inflicted by these ubiquitous lesions and of their involvement in the flux of DNA metabolism. We present a proteomics-based strategy for assembling at least a partial catalogue of proteins capable of binding AP sites in DNA. The general scheme relies on the sensitivity of many AP site-bound protein species to NaBH(4) cross-linking. An affinity-tagged substrate is used to facilitate isolation of the cross-linked species, which are then separated and analyzed by mass spectrometry methods. We report identification of seven proteins from Escherichia coli (AroF, DnaK, MutM, PolA, TnaA, TufA, and UvrA) and two proteins from bakers' yeast (ARC1 and Ygl245wp) reactive for AP sites in this system.
Rieger
Robert A
RA
Department of Pharmacology, Stony Brook University, Stony Brook, NY 11794, USA.
Zaika
Elena I
EI
Xie
Weiping
W
Johnson
Francis
F
Grollman
Arthur P
AP
Iden
Charles R
CR
Zharkov
Dmitry O
DO
D002457:Cell Extracts; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D040901:Proteomics; D012441:Saccharomyces cerevisiae; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019809:Streptavidin
D002457:Cell Extracts; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D004247:DNA; D019809:Streptavidin