TY - JOUR
T1 - DNA ligase IV and XRCC4 form a stable mixed tetramer that functions synergistically with other repair factors in a cell-free end-joining system
AU - Lee, Kyung Jong
AU - Huang, Juren
AU - Takeda, Yoshihiko
AU - Dynan, William S.
PY - 2000/11/3
Y1 - 2000/11/3
N2 - Repair of DNA double-strand breaks in mammalian cells occurs via a direct nonhomologous end-joining pathway. Although this pathway can be studied in viva 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.
AB - Repair of DNA double-strand breaks in mammalian cells occurs via a direct nonhomologous end-joining pathway. Although this pathway can be studied in viva 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.
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U2 - 10.1074/jbc.M004011200
DO - 10.1074/jbc.M004011200
M3 - Article
C2 - 10945980
AN - SCOPUS:0034602285
SN - 0021-9258
VL - 275
SP - 34787
EP - 34796
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 44
ER -