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

Jae Wan Lee, Luis Blanco, Tong Zhou, Miguel Garcia-Diaz, Katarzyna Bebenek, Thomas A. Kunkel, Zhigang Wang, Lawrence F. Povirk

Research output: Contribution to journalArticlepeer-review

190 Scopus citations

Abstract

Accurate repair of free radical-mediated DNA double-strand breaks by the nonhomologous end joining pathway requires replacement of fragmented nucleotides in the aligned ends by a gap-filling DNA polymerase. Nuclear extracts of human HeLa cells, supplemented with recombinant XRCC4-DNA ligase IV complex (XRCC4/ligase 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 candidate enzymes, DNA polymerases μ and λ both of which were present in the extracts. For a DNA substrate with partially complementary 3′ overhangs and a 2-base gap, antibodies to polymerase λ completely eliminated both gap filling and accurate end joining, whereas antibodies to polymerase μ had little effect. Immunodepletion of polymerase λ but not polymerase μ likewise blocked both gap filling and end joining, and both functions could be restored by addition of recombinant polymerase λ. Recombinant polymerase μ and a truncated polymerase λ lacking the Brca1 C-terminal domain, were at least 10-fold less active in restoring gap filling to the immunodepleted extracts, and polymerase β was completely inactive. The results suggest that polymerase A is the primary gap-filling polymerase for accurate nonhomologous end joining, and that the Brca1 C-terminal domain is required for this activity.

Original languageEnglish
Pages (from-to)805-811
Number of pages7
JournalJournal of Biological Chemistry
Volume279
Issue number1
DOIs
StatePublished - Jan 2 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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