Microsoft Word - 2013 manuscript.55.docx

Nonhomologous end joining repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. Ku, XRCC4/Ligase IV (XL) and XLF have a remarkable mismatched end (MEnd) ligase activity, particularly for ends with mismatched 3' overhangs, but the mechanism has remained obscure. Here, we showed XL required Ku to bind DNA, while XLF required both Ku and XL to bind DNA. We detected cooperative assembly of one or two Ku molecules and up to 5 molecules each of XL and XLF into a Ku:XL:XLF-DNA (MEnd ligase-DNA) complex. XLF mutations that disrupted its interactions with XRCC4 or DNA also disrupted complex assembly and end joining. Together with published co-crystal structures of truncated XRCC4 and XLF proteins, our data with full-length Ku, XL and XLF bound to DNA indicate assembly of a filament containing Ku plus alternating XL and XLF molecules. By contrast, in the absence of XLF, we detected cooperative assembly of up to 6 molecules each of Ku and XL into a Ku:XL-DNA complex, consistent with a filament containing alternating Ku and XL molecules. Despite a lower molecular weight, MEnd ligase-DNA had a lower electrophoretic mobility than Ku:XLDNA. The anomalous difference in mobility and difference in XL to Ku molar ratio suggest that MEnd ligase-DNA has a distinct structure that successfully aligns mismatched DNA ends for ligation. Nonhomologous end joining (NHEJ) repairs DNA double-strand breaks created by exogenous agents such as ionizing radiation, or by the endogenous mechanism for generating immunological diversity, V(D)J recombination (1,2). NHEJ recognizes broken ends, brings them into juxtaposition, and joins the ends while optimizing the preservation of DNA sequence. NHEJ even preserves 3’-overhanging DNA sequences created during V(D)J recombination by hairpin opening (P-nucleotide addition) or by terminal deoxynucleotidyl transferase activity (Nnucleotide addition). Thus, NHEJ has a mechanism for joining DNA ends with mismatched 3’ overhangs. Core proteins for NHEJ include Ku, DNAdependent protein kinase catalytic subunit (DNAPKcs), XRCC4/Ligase IV (XL) and XRCC4-like factor (XLF, also known as Cernunnos). Ku, which consists of two subunits, Ku70 and Ku80, binds DNA ends with high affinity (3,4) and then recruits DNA-PKcs to the ends (5). DNA-PKcs brings the ends together into a synaptic complex and then undergoes activation of its kinase domain http://www.jbc.org/cgi/doi/10.1074/jbc.M113.464115 The latest version is at JBC Papers in Press. Published on April 25, 2013 as Manuscript M113.464115