The results summarized inSupplementary Figure S2Cshow no increase in the number of foci per cell between wt,LIG1/andLIG1/LIG4/cells
The results summarized inSupplementary Figure S2Cshow no increase in the number of foci per cell between wt,LIG1/andLIG1/LIG4/cells. cell line with mutatedLIG1alleles (6,7), 46BR, ERK2 from an immunodeficient patient, shows defects in Okazaki fragment joining but nearly normal overall growth characteristics. ALIG1knockout mouse harboring homozygous deletions of the 3-end of theLIG1gene is embryonic lethal (8). However, mouse embryonic fibroblast (MEF) cell lines generated from such embryos show defects in Okazaki fragment joining but normal proliferation CCR4 antagonist 2 patterns (8,9). Therefore, the essential role of LigI in DNA replication is still under debate. The second family of ligases found in all eukaryotes is that of LigIV. The main function of LigIV is the ligation step during the repair of DNA double strand breaks (DSBs) by the canonical non-homologous end-joining (NHEJ) pathway. Through breast cancer gene 1 carboxy terminal (BRCT) motifs, LigIV interacts with X-ray cross complementing 4 (Xrcc4) and CCR4 antagonist 2 becomes integrated in a NHEJ pathway (to be termed here D-NHEJ), which in vertebrates also comprises XRCC4-like factor (XLF), and the DNA-dependent protein kinase (DNA-PK) complex, consisting of the Ku heterodimer and the catalytic subunit, DNA-PKcs (10,11). Although in the mouseLIG4knockout is embryonic lethal (1214), this lethality can be rescued by concomitant loss of p53 function andLIG4//p53/MEFs are highly radiosensitive and show defects in D-NHEJ (1416). The LigIII family is newer evolutionary and restricted to vertebrates (2). Nuclear and mitochondrial versions of LigIII are ubiquitously synthesized fromLIG3mRNA by an alternative translation initiation mechanism (17,18). In addition, germ-cell-specific alternative splicing of the LigIII 3-coding exon generates LigIII (18,19). Nuclear LigIII interacts with Xrcc1 and functions in the short-patch subpathway of BER (20), the repair of single strand breaks (21,22) and a CCR4 antagonist 2 NER subpathway (23). There is also evidence that LigIII is a component of an alternative pathway of NHEJ functioning as a backup (B-NHEJ) to D-NHEJ (24,25). Deletion ofLIG3has consequences significantly more severe than deletion of eitherLIG1orLIG4and attempts to generateLIG3knockout cells or animals had failed until recently (26). Recent work shows that loss of mitochondria ligase function underlies this lethality and that viability of aLIG3knockout is rescued by other eukaryotic ligases targeted to this organelle, or CCR4 antagonist 2 by expressing prokaryotic homologs (27,28). Although these observations indicate thatLIG3is dispensable, they leave open the question as to whether LigI or LigIV substitute for important LigIII functions, and vice-versa, as a result of unknown functional redundancy among eukaryotic DNA ligases. Here, we employ the chicken B cell line, DT40, and powerful conditional targeting approaches to investigate the role of the different DNA ligases in DNA replication and to study the inherent functional flexibility built by evolution into the vertebrate ligase system. == MATERIAL AND METHODS == == Cell culture and electroporation == DT40 cells were grown at 41C in a mixture of D-MEM/F12 growth medium supplemented with 10% fetal bovine serum, 1% chicken serum, 50 M -mercaptoethanol CCR4 antagonist 2 in a humidified incubator supplemented with 5% CO2. All cells were routinely maintained in the logarithmic phase of growth. Stable transfectants were selected in 15 g/ml of blasticidin S, 1 g/ml mycophenolic acid or 1 g/ml of puromycin, as appropriate. Targeted clones were screened by polymerase chain reaction (PCR) according to Arakawaet al.(29). Two methods of electroporation were used to introduce DNA into DT40 cells. In the first protocol, electroporation was carried out with cells suspended in complete growth medium, and 107cells were electroporated using the GenePulser-Xcell (BIORAD).
