DNA Polymerase β and Flap Endonuclease 1 Enzymatic Specificities Sustain DNA Synthesis for Long Patch Base Excision Repair

• Published in: The Journal of biological chemistry 2005-02, Vol.280 (5), p.3665
• Main Authors: Yuan Liu, William A. Beard, David D. Shock, Rajendra Prasad, Esther W. Hou, Samuel H. Wilson
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M412922200

DNA polymerase β (pol β) and flap endonuclease 1 (FEN1) are key players in pol β-mediated long-patch base excision repair (LP-BER). It was proposed that this type of LP-BER is accomplished through FEN1 removal of a 2- to 11-nucleotide flap created by pol β strand displacement DNA synthesis. To understand how these enzymes might cooperate during LP-BER, we characterized purified human pol β DNA synthesis by utilizing various BER intermediates, including single-nucleotide-gapped DNA, nicked DNA, and nicked DNA with various lengths of flaps all with a 5′-terminal tetrahydrofuran (THF) residue. We observed that nicked DNA and nicked-THF flap DNA were poor substrates for pol β-mediated DNA synthesis; yet, DNA synthesis was strongly stimulated by purified human FEN1. FEN1 did not improve pol β substrate binding. FEN1 cleavage activity was required for the stimulation, suggesting that FEN1 removed a barrier to pol β DNA synthesis. In addition, FEN1 cleavage on both nicked and nicked-THF flap DNA resulted in a one-nucleotide gapped DNA molecule that was an ideal substrate for pol β. This study demonstrates that pol β cooperates with FEN1 to remove DNA damage via a “Hit and Run” mechanism, involving alternating short gap production by FEN1 and gap filling by pol β, rather than through coordinated formation and removal of a strand-displaced flap.