Abstract
Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. To repair the broken ends, NHEJ processes noncompatible ends into a ligatable form but suppresses processing of compatible ends. It is not known how NHEJ controls polymerase and nuclease activities to act exclusively on noncompatible ends. Here, we analyzed processing independently of ligation by using a two-stage assay with extracts that recapitulated the properties of NHEJ in vivo. Processing of noncompatible ends required wortmannin-sensitive kinase activity. Since DNA-dependent protein kinase catalytic subunit (DNA-PKcs) brings the ends together before undergoing activation of its kinase, this suggests that processing occurred after synapsis of the ends. Surprisingly, all polymerase and most nuclease activity required XRCC4/Ligase IV. This suggests a mechanism for how NHEJ suppresses processing to optimize the preservation of DNA sequence.
Highlights
Processing of DNA ends in vivo has been studied in the context of V(D)J recombination
XRCC4/Ligase IV (XL) interacts with polymerase , polymerase , and Cernunnos/XLF [6, 7], but it is not known whether XL has a function other than ligation
Joining requires Ku, XL, and DNA-PKcs. It is not known whether XL or DNAPKcs participates in the processing step or the ligation step of Nonhomologous end-joining (NHEJ)
Summary
Processing of DNA ends in vivo has been studied in the context of V(D)J recombination. Extracts from HeLa and lymphoblastoid cells processed noncompatible ends using polymerase and nuclease activities (Fig. 2 and supplemental Fig. 4). HeLa extract with added dNTPs joined EcoRI-KpnI ends with a high efficiency of 6.38%, and 10 of 20 strands were processed by nucleotide addition (Fig. 2).
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