Role of DNA polymerase lambda, XRCC4, and DNA ligase IV in alignment-based gap-filling of nonhomologous end joining

DNA double strand breaks (DSB) are the most destructive type of DNA damage that interferes with normal cellular function and survival. Exposure to ionizing radiation (IR) and radiomimetic agents result in DSBs with mostly 3 ' phosphoglycolate- (PG) ends and with missing nucleotides. X-ray cross complementing factor 4 and DNA ligase IV (X4L4) are among many proteins involved in nonhomologous end joining (NHEJ) that repair these DSBs, and their presence has been found to be critical for cells' survival when they are exposed to IR. The role of X4L4 in coordinating gap-filling of missing nucleotides at DSB termini in conjunction with the identity of the DNA polymerase (pol) involved has never been definitively determined. Thus, to determine whether DNA pol requires the presence of X4L4 during the gap-filling stage of NHEJ, end-joining assays were performed in whole cell extracts of normal Chinese hamster ovary cells (CHO-K1) and XR-1 cells, deficient in X4L4. The radiolabeled DNA substrates used in the assay had partially cohesive PG- or hydroxyl-terminated 3' overhangs with a gap in each strand, such that addition of a nonligatable dideoxynucleotide (ddTTP) could interrupt the end-joining process at the gap-filling step. The results obtained with CHO-K1 cell extracts revealed competency in repairing and gap-filling. XR-1 extracts, however, did not have any end-joining activities as predicted. Surprisingly, gap-filling activities in XR-1 extracts could not be detected by the addition of ddTTP. However, complementation of recombinant X4L4 (gift of S. M. Yannone & D. J. Chen) restored both end-joining and gap-filling activities in XR-1 extracts suggesting a requirement for X4L4 in gap-filling by the DNA pol. Second, to determine the identity of the DNA pol that catalyzes this gap-filling, an antibody inhibition assay and an immunodepletion assay were used with antibodies raised against pol mu or pol lambda in Hela nuclear cell extracts. Antibody against pol lambda completely blocked gap-filling activity while pol mu antibody had a negligible effect. Also, in lambda-depleted extracts complemented with recombinant pol lambda, the gap-filling activity could be fully restored. This strongly supports our hypothesis that pol lambda may be the gap-filling DNA pol in NHEJ.