title: Pol β associated complex and base excision repair factors in mouse fibroblasts.
abstract: During mammalian base excision repair (BER) of lesion-containing DNA, it is proposed that toxic strand-break intermediates generated throughout the pathway are sequestered and passed from one step to the next until repair is complete. This stepwise process is termed substrate channeling. A working model evaluated here is that a complex of BER factors may facilitate the BER process. FLAG-tagged DNA polymerase (pol) β was expressed in mouse fibroblasts carrying a deletion in the endogenous pol β gene, and the cell extract was subjected to an 'affinity-capture' procedure using anti-FLAG antibody. The pol β affinity-capture fraction (ACF) was found to contain several BER factors including polymerase-1, X-ray cross-complementing factor1-DNA ligase III and enzymes involved in processing 3'-blocked ends of BER intermediates, e.g. polynucleotide kinase and tyrosyl-DNA phosphodiesterase 1. In contrast, DNA glycosylases, apurinic/aprymidinic endonuclease 1 and flap endonuclease 1 and several other factors involved in BER were not present. Some of the BER factors in the pol β ACF were in a multi-protein complex as observed by sucrose gradient centrifugation. The pol β ACF was capable of substrate channeling for steps in vitro BER and was proficient in in vitro repair of substrates mimicking a 3'-blocked topoisomerase I covalent intermediate or an oxidative stress-induced 3'-blocked intermediate.
authors.affiliation: Laboratory of Structural Biology, NIEHS, National Institutes of Health, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Prasad
authors.forename: Jason G
authors.initials: JG
authors.lastname: Williams
authors.forename: Esther W
authors.initials: EW
authors.lastname: Hou
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
mesh_terms: D000818:Animals; D002457:Cell Extracts; D004247:DNA / Q000378:metabolism; D059018:DNA Contamination; D019951:DNA Polymerase beta / Q000378:metabolism*; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000302:isolation & purification / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D051379:Mice; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000302:isolation & purification
chemical_list: D002457:Cell Extracts; D004247:DNA; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D019951:DNA Polymerase beta; D045643:DNA Repair Enzymes