DNA ligase IV and XRCC4 form a stable mixed tetramer that functions synergistically with other repair factors in a cell-free end-joining system.
Repair of DNA double-strand breaks in mammalian cells occurs via a direct nonhomologous end-joining pathway. Although this pathway can be studied in vivo and in crude cell-free systems, a deeper understanding of the mechanism requires reconstitution with purified enzymes. We have expressed and purified a complex of two proteins that are critical for double-strand break repair, DNA ligase IV (DNL IV) and XRCC4. The complex is homogeneous, with a molecular mass of about 300,000 Da, suggestive of a mixed tetramer containing two copies of each polypeptide. The presence of multiple copies of DNL IV was confirmed in an experiment where different epitope-tagged forms of DNL IV were recovered simultaneously in the same complex. Cross-linking suggests that an XRCC4.XRCC4 dimer interface forms the core of the tetramer, and that the DNL IV polypeptides are in contact with XRCC4 but not with one another. Purified DNL IV.XRCC4 complex functioned synergistically with Ku protein, the DNA-dependent protein kinase catalytic subunit, and other repair factors in a cell-free end-joining assay. We suggest that a dyad-symmetric DNL IV.XRCC4 tetramer bridges the two ends of the broken DNA and catalyzes the coordinate ligation of the two DNA strands.
Lee
K J
KJ
Gene Regulation Program, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA.
Huang
J
J
Takeda
Y
Y
Dynan
W S
WS
D000818:Animals; D001483:Base Sequence; D001704:Biopolymers; D002474:Cell-Free System; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008970:Molecular Weight; D011233:Precipitin Tests; D018411:Spodoptera
D001704:Biopolymers; D017931:DNA Primers; D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; C101467:XRCC4 protein, human; D011088:DNA Ligases; D000072481:DNA Ligase ATP