Requirement for XRCC4 and DNA ligase IV in alignment-based gap filling for nonhomologous DNA end joining in vitro.
In the nonhomologous end joining pathway of DNA double-strand break repair, the ligation step is catalyzed by a complex of XRCC4 and DNA ligase IV. Extracts of CHO-K1 cells are able to accurately rejoin a site-specific free radical-mediated double-strand break with partially complementary overhangs, by a mechanism involving alignment-based gap filling followed by ligation. Extracts of XR-1 cells, which lack XRCC4 and DNA ligase IV, carried out neither gap filling nor ligation. Supplementation of the extracts with recombinant XRCC4/ligase IV, but not with XRCC4 alone, restored gap filling and accurate end joining. The results imply that XRCC4 and ligase IV are essential for alignment-based gap filling, as well as for final ligation of the breaks.
Lee
Jae Wan
JW
Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Yannone
Steven M
SM
Chen
David J
DJ
Povirk
Lawrence F
LF
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection
D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; D011994:Recombinant Proteins; C101467:XRCC4 protein, human; D011088:DNA Ligases; D000072481:DNA Ligase ATP