Implication of DNA Polymerase in Alignment-based Gap Filling for Nonhomologous DNA End Joining in Human Nuclear Extracts*

Accurate repair of free radical-mediated DNA double-strand breaks by the nonhomologous end joining path-way requires replacement of fragmented nucleotides in the aligned ends by a gap-filling DNA polymerase. Nu-clear extracts of human HeLa cells, supplemented with recombinant XRCC4-DNA ligase IV complex (XRCC4/li-gase IV), were capable of accurately rejoining model double-strand break substrates with a 1- or 2-base gap, and the gap-filling step was dependent on XRCC4/ligase IV. To determine what polymerase was responsible for gap filling, end joining was examined in the presence of polyclonal antibodies against each of two prime candi-date enzymes, DNA polymerases and , both of which were present in the extracts. For a DNA substrate with partially complementary 3 overhangs and a 2-base gap