title: Enzymatic Activity Assays for Base Excision Repair Enzymes in Cell Extracts from Vertebrate Cells.
abstract: We previously reported enzymatic activity assays for the base excision repair (BER) enzymes DNA polymerase β (pol β), aprataxin (APTX), and flap endonuclease 1 (FEN1) in cell extracts fromSaccharomyces cerevisiae(Çağlayan and Wilson, 2014). Here, we describe a method to prepare cell extracts from vertebrate cells to investigate these enzymatic activities for the processing of the 5'-adenylated-sugar phosphate-containing BER intermediate. This new protocol complements our previous publication. The cell lines used are wild-type and APTX-deficient human lymphoblast cells from an Ataxia with Oculomotor Apraxia Type 1 (AOA1) disease patient, wild-type and APTX-null DT40 chicken B cells, and mouse embryonic fibroblast (MEF) cells. This protocol is a quick and efficient way to make vertebrate cell extracts without using commercial kits.
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
authors.forename: Melike
authors.initials: M
authors.lastname: Çağlayan
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
authors.forename: Julie K
authors.initials: JK
authors.lastname: Horton
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson