The 9-1-1 complex has no effect on the activities of Fen 1 and Lig I in long patch base excision repair

Abstract

<p><b>Copyright information:</b></p><p>Taken from "The checkpoint clamp, Rad9-Rad1-Hus1 complex, preferentially stimulates the activity of apurinic/apyrimidinic endonuclease 1 and DNA polymerase β in long patch base excision repair"</p><p></p><p>Nucleic Acids Research 2007;35(8):2596-2608.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885638.</p><p>© 2007 The Author(s)</p> The LP-BER reaction was performed as described in ‘Materials and Methods’. Reactions were stopped by adding an equal volume of formamide-dye solution and products were analyzed on a 10% denaturing polyacrylamide gel. The reaction mixtures (10 μl) contained (besides all components described in Materials and Methods) P-5′-labeled 100 bp duplex oligonucleotide (50 fmol), APE 1 (55 fmol), Pol β (64 fmol), Fen 1 (9 fmol), and of Lig I (245 fmol). Reactions were incubated for 20 min at 37°C with the indicated amounts of the 9-1-1 complex. The reaction mixtures (10 μl) contained (besides all components described in Materials and Methods) P-5′-labeled 100 bp duplex oligonucleotide (50 fmol), APE 1 (55 fmol), Pol β (64 fmol), Fen 1 (93 fmol), and Lig I (25 fmol). Reactions were incubated for 20 min at 37°C with the indicated amounts of the 9-1-1 complex. A Fen 1 reaction mixture (10 μl) contained (besides all components described in Materials and Methods) a 10 nucleotide flap substrate (50 fmol, see B) and Fen 1 (25 fmol). Reactions were incubated for 20 min at 37°C with the indicated amounts of the 9-1-1 complex. A Lig I reaction mixture (10 μl) contained (besides all components described in Materials and Methods) a nicked oligonucleotide (50 fmol, see B) and Lig I (0.5 fmol). Reactions were incubated for 20 min at 37°C with the indicated amounts of the 9-1-1 complex