Nucleic Acids Research Advance Access published online on May 6, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp283
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Nucleic Acid Enzymes |
Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases 
and µ for nonhomologous end joining in human whole-cell extracts
1Department of Pharmacology and Toxicology, 2Department of Radiation Oncology, Virginia Commonwealth University, Richmond, VA 23298, USA, 3Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1, 4Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA and 5Institut National de la Santé et de la Recherche Médical, Paris F-75015, France
*To whom correspondence should be addressed. Tel: +1 804 828 9640; Fax: +1 804 827 0635; Email: lpovirk{at}vcu.edu
Received August 31, 2008. Revised April 11, 2009. Accepted April 13, 2009.
XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5' or 3' overhangs, and no joining at all of partially complementary 3' overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped using dideoxynucleotides revealed that there was no gap filling on aligned DSB ends in the Cernunnos-deficient extracts. Recombinant Cernunnos protein restored gap filling and end joining of partially complementary overhangs, and stimulated joining of cohesive ends more than twentyfold. XLF-dependent gap filling was nearly eliminated by immunodepletion of DNA polymerase 
, but was restored by addition of either polymerase or polymerase µ. Thus, Cernunnos is essential for gap filling by either polymerase during nonhomologous end joining, suggesting that it plays a major role in aligning the two DNA ends in the repair complex.