Simsek D , Brunet E , Wong SY , Katyal S , Gao Y , McKinnon PJ , Lou J , Zhang L , Li J , Rebar EJ , Gregory PD , Holmes MC , Jasin M .

R56 NS037956 NINDS NIH HHS , GM54668 NIGMS NIH HHS , R01 NS037956 NINDS NIH HHS , CA21765 NCI NIH HHS , NS37956 NINDS NIH HHS , P30 CA021765 NCI NIH HHS , R01 GM054668 NIGMS NIH HHS , NIHGM54668 PHS HHS , R01 NS037956-09 NINDS NIH HHS

	Figure 1. Lig3-deficient mouse cells have fewer chromosomal translocations and reduced microhomology at junctions.(A) DNA ligases expressed from transgenes in Lig3KO/KO cells. Wild-type Lig3 is expressed in control cells. Nuclear Lig3-deficient cells express mitochondria-restricted Lig3 (MtLig3-ΔBRCT-GFP-NES), while Lig3 null cells express mitochondria-targeted Lig1 (MtLig1 or MtLig1-ΔNLS). All transgenes are GFP tagged. MLS, mitochondrial leader sequence from Lig3 for mitochondrial localization; ZnF, zinc finger domain; BRCT, BRCA1 C-terminal related domain; DBD, DNA-binding domain; CC, catalytic core; *M88T, mutation of the Lig3 nuclear translation initiation site which disrupts translation of a nuclear-specific form of Lig3; NES, nuclear export signal derived from MAPKK to exclude Lig3 from the nucleus; ΔNLS, deletion of Lig1 nuclear localization signal (amino acids 135 to 147) to decrease nuclear localization of Lig1; ΔBRCT, deletion of theLig3 BRCT domain (amino acids 934 to 1009); GFP, green fluorescent protein. Triangles denote position of deletions of the indicated domains. A color scheme is used throughout the figures to assist in tracking various ligase forms (gray, wild-type Lig3; purple, mitochondria-only Lig3; orange, Lig1 forms). (B) Translocations are reduced in cell lines without nuclear Lig3. By contrast, translocations increase in cells deficient for Lig4–XRCC4 complex. (C) Microhomology length distributions for der(6) breakpoint junctions are similar for control cells, cells depleted for Lig1, and cells deficient for Lig4–XRCC4, and differ from that expected by the chance presence of microhomology. Only junctions with simple deletions (i.e., without an insertion) are included. The probability that a junction will have X nucleotides of microhomology by chance assumes an unbiased base composition and is calculated as previously described [9]. The transgenes present in the Lig3KO/KO cells are indicated in italics. (D) Microhomology length distributions in cells without nuclear Lig3 are shifted to a distribution similar to that expected by chance.
	Figure 2. Induction of chromosomal translocations at endogenous mouse loci.(A) DSBs are induced at cleavage sites for the zinc finger nucleases (ZFNs) ZFNRosa26 and ZFNH3f3b on chromosomes 6 and 11, respectively. Joining of DNA ends from 2 different chromosomes can lead to the translocations generating derivative chromosomes der(6) and der(11). Sequences surrounding the DSBs generated by the ZFNs, together with sequences of the resultant translocation chromosomes, are shown. The latter assumes fill-in of the 5′ overhangs and no loss of sequence information from the DNA ends. (B) Nested PCR is used to identify translocation breakpoint junctions for der(6) and der(11), as shown, which is dependent on expression of both ZFNRosa26 and ZFNH3f3b.
	Figure 3. Deletion lengths for der(6) breakpoint junctions.Deletion lengths for the indicated genotypes do not differ significantly from each other. Each value represents the combined deletion from both ends of an individual junction. The median deletion length for each genotype is indicated by a bar on the graph and the value is given below the graph.
	Figure 4. Lig1, but not Lig4, is a backup DNA ligase for translocation formation.Lig1 depletion results in reduced translocation frequency in nuclear Lig3-deficient cells (Lig3KO/KO; MtLig3 ΔBRCT GFP NES), but not in control cells expressing wild-type Lig3 (Lig3KO/KO; Lig3 GFP). By contrast, Lig4 depletion increases translocations significantly in wild-type cells but not nuclear Lig3-deficient cells. Lig1 and Lig4 lentiviral shRNA knockdown are similar in both nuclear Lig3-deficient cells and cells expressing wild-type Lig3. scr, shRNA with a scrambled sequence.
	Figure 5. Analysis of Lig3 domain requirements in translocation formation.(A) Deletion of the Lig3 BRCT domain has no effect on translocation frequency, while deletion of the Lig3 ZnF domain reduces translocations. Triangles denote the deletions of the indicated domains. ΔZnF, deletion of Lig3 amino acids 89 to 258 including the ZnF itself and additional sequences which corresponds to a previously described Lig3 ΔZnF mutant [37], [38]; ΔBRCT, deletion of Lig3 amino acids 934 to 1009. (B) Microhomology length distributions in translocation junctions are similar in cells expressing wild-type Lig3 and those expressing either ZnF or BRCT deletions of Lig3. (C) Large, complex insertions are more frequent in cells expressing Lig3 deleted for the ZnF domain. The summary “All Genotypes – Lig3 ΔZnF” denotes all junctions analyzed except those from Lig3KO/KO; Lig3 ΔZnF cells. P values are calculated relative to this group with a Fisher's exact test.
	Figure 6. Model for the role of the three mammalian DNA ligases in chromosomal translocation formation in mouse cells.Upon DSB formation on two chromosomes (blue and red lines), Lig4 promotes intrachromosomal joining to maintain genomic integrity and suppresses translocations. Translocations are formed by Lig3, preferentially at short microhomologies (green lines). DNA end resection, which is likely suppressed by Lig4, forms single-stranded DNA to expose microhomologies which can anneal. In the absence of Lig3, Lig1 can form translocations independent of pre-existing microhomology. However, microhomology can also be generated by polymerase (Pol) activity at a DNA end.

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