The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous DNA recombination (HR) in Saccharomyces cerevisiae and is essential for the proliferation of vertebrate cells. Five vertebrate genes, RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on the basis of their sequence similarity to Rad51 (Rad51 paralogs). We generated mutants deficient in each of these proteins in the chicken B-lymphocyte DT40 cell line and report here the comparison of four new mutants and their complemented derivatives with our previously reported rad51b mutant. The Rad51 paralog mutations all impair HR, as measured by targeted integration and sister chromatid exchange. Remarkably, the mutant cell lines all exhibit very similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents (mitomycin C and cisplatin), mild sensitivity to gamma rays, and significantly attenuated Rad51 focus formation during recombinational repair after exposure to gamma rays. Moreover, all mutants show partial correction of resistance to DNA damage by overexpression of human Rad51. We conclude that the Rad51 paralogs participate in repair as a functional unit that facilitates the action of Rad51 in HR.Double-strand DNA breaks (DSBs) are produced by ionizing radiation (IR) and certain chemicals, and they likely occur frequently during DNA replication (21, 34). A single unrepaired DSB may stimulate cell cycle checkpoints and cause cell death (3, 25). Homologous recombination (HR) has emerged as a major DSB repair pathway in mammalian cells (29,35,44,65,66), as well as in the yeast Saccharomyces cerevisiae. Indeed, the analysis of radiosensitive yeast mutants has revealed a number of key genes involved in HR, which comprise the RAD52 epistasis group (2,32,54), and the HR pathway is conserved from yeast to humans (4,18,53,65). Although yeast is capable of proliferating at a reduced rate in the absence of functional HR, this repair pathway is essential for viability in cycling vertebrate cells for coping with DNA lesions arising during DNA replication (55,56,67,73). This species difference is probably due to the several-hundred-fold difference in genome size between vertebrates and yeast.ScRad51 is closely related to the Escherichia coli recombination protein RecA (5). Among the proteins of the Rad52 epistasis group, Rad51 has the highest degree of structural and functional conservation among all eukaryotes. The high degree of identity of ScRad51 with the human homolog (59% identity) and chicken homolog (59% identity) suggests that Rad51's function is conserved across eukaryotes. A central role for Rad51 in HR in vertebrates is supported by the finding that Rad51 deficiency (36, 55, 67), but not Rad52 or Rad54 deficiency, is lethal to cells (4,18,49,72). In vitro studies show that RecA and Rad51 form multimeric helical nucleoprotein filaments that are assembled on single-stranded DNA (ssDNA) (2). Recent work suggests that the preferred DNA substrat...

The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/ RAD51L1, a member of the Rad51 family, knocked out. RAD51B؊/؊ cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle. Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus. RAD51B؊/؊ cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to ␥-rays. The formation of damage-induced Rad51 nuclear foci is much reduced in RAD51B ؊/؊ cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR. These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity. Double-strand DNA breaks (DSBs) occur during DNA replication and are produced by ionizing radiation. Since DSBs are so deleterious to the cell, it is not surprising that there are two DSB repair pathways: nonhomologous end joining (NHEJ) and homologous recombination repair (HRR). Repair of DSBs by HRR requires the presence of homologous duplex DNA elsewhere in the genome, i.e., either a homologous chromosome or, more likely, a sister chromatid. NHEJ simply acts to process and ligate broken ends without a requirement for extensive homology. These pathways are conserved from the yeast Saccharomyces cerevisiae to humans (5,8,9,19,49,53,64). While HRR is the primary mechanism of DSB repair in yeast, vertebrate cells use both the NHEJ and HRR pathways extensively (28,34,35,44). The analysis of radiosensitive yeast mutants has revealed a number of genes involved in HRR, which comprise the RAD52 epistasis group (reviewed in references 4, 29, and 51).Among the members of the RAD52 epistasis group, the structure and function of Rad51 have been conserved to a remarkable degree among all eukaryotes. Rad51 is structurally and functionally related to the Escherichia coli recombination protein RecA (reviewed in reference 32). The functional forms of both RecA and Rad51 are multimeric helical nucleoprotein filaments that form on single-stranded DNA ends produced at DSBs (41). These filaments are involved in the search for homologous sequence, DNA pairing, and strand exchange. Recombination intermediates produced in this way are then processed further in reactions that involve DNA synthesis, branch migration, resolution of Holliday junctions, and ligation (reviewed in reference 4). The conservation of the RAD52 epistasis group genes from yeast to vertebrate cells suggests that the basic mechanism of HRR is maintained during evolution. Howe...

Parp-1 and Parp-2 are activated by DNA breaks and have been implicated in the repair of DNA single-strand breaks (SSB). Their involvement in double-strand break (DSB) repair mediated by homologous recombination (HR) or nonhomologous end joining (NHEJ) remains unclear. We addressed this question using chicken DT40 cells, which have the advantage of carrying only a PARP-1 gene but not a PARP-2 gene. We found that PARP-1 À/À DT40 mutants show reduced levels of HR and are sensitive to various DSB-inducing genotoxic agents. Surprisingly, this phenotype was strictly dependent on the presence of Ku, a DSBbinding factor that mediates NHEJ. PARP-1/KU70 double mutants were proficient in the execution of HR and displayed elevated resistance to DSB-inducing drugs. Moreover, we found deletion of Ligase IV, another NHEJ gene, suppressed the camptothecin of PARP-1 À/À cells. Our results suggest a new critical function for Parp in minimizing the suppressive effects of Ku and the NHEJ pathway on HR.

Cross-linking agents that induce DNA interstrand cross-links (ICL) are widely used in anticancer chemotherapy. Yeast genetic studies show that nucleotide excision repair (NER), Rad6/Rad18-dependent postreplication repair, homologous recombination, and cell cycle checkpoint pathway are involved in ICL repair. To study the contribution of DNA damage response pathways in tolerance to cross-linking agents in vertebrates, we made a panel of gene-disrupted clones from chicken DT40 cells, each defective in a particular DNA repair or checkpoint pathway, and measured the sensitivities to cross-linking agents, including cis-diamminedichloroplatinum (II) (cisplatin), mitomycin C, and melphalan. We found that cells harboring defects in translesion DNA synthesis (TLS), Fanconi anemia complementation groups (FANC), or homologous recombination displayed marked hypersensitivity to all the cross-linking agents, whereas NER seemed to play only a minor role. This effect of replicationdependent repair pathways is distinctively different from the situation in yeast, where NER seems to play a major role in dealing with ICL. Cells deficient in Rev3, the catalytic subunit of TLS polymerase PolZ, showed the highest sensitivity to cisplatin followed by fanc-c. Furthermore, epistasis analysis revealed that these two mutants work in the same pathway. Our genetic comprehensive study reveals a critical role for DNA repair pathways that release DNA replication block at ICLs in cellular tolerance to cross-linking agents and could be directly exploited in designing an effective chemotherapy. (Cancer Res 2005; 65(24): 11704-11)

Systemic cytokine activity in response to Toll-like receptor (TLR) signaling induces the expression of various proteins in the liver after infections. Here we show that Interleukin-7 (IL-7), the production of which was thought to occur at a constant rate in vivo, was a hepatically expressed protein that directly controled T cell responses. Depletion of IL-7 expression in the liver abrogated several TLR-mediated T cell events, including enhanced CD4+ T cell and CD8+ T cell survival, augmented CD8+ T cell cytotoxic activity, and the development of experimental autoimmune encephalitis, a Th17 cell-mediated autoimmune disease. Thus, T cell responses are regulated by hepatocyte-derived IL-7, which is expressed in response to TLR signaling in vivo. We suggested that TLR-induced IL-7 expression in the liver, which is an acute-phase response, may be a good diagnostic and therapeutic target for efficient vaccine developments and for conditions characterized by TLR-mediated T cell dysregulation, including autoimmune diseases.

Clinical topoisomerase I (Top1) and II (Top2) inhibitors trap topoisomerases on DNA, thereby inducing protein-linked DNA breaks. Cancer cells resist the drugs by removing topoisomerase-DNA complexes, and repairing the drug-induced DNA double-strand breaks (DSBs) by homologous recombination (HR) and non-homologous end-joining (NHEJ). Because numerous enzymes and cofactors are involved in the removal of the topoisomerase-DNA complexes and DSB repair, it has been challenging to comprehensively analyze the relative contribution of multiple genetic pathways in vertebrate cells. Comprehending the relative contribution of individual repair factors would give insights into the lesions induced by the inhibitors and genetic determinants of response. Ultimately, this information would be useful to target specific pathways to augment the therapeutic activity of topoisomerase inhibitors. To this end, we put together 48 isogenic DT40 mutant cells deficient in DNA repair and generated one cell line deficient in autophagy (ATG5). Sensitivity profiles were established for three clinically relevant Top1 inhibitors (camptothecin and the indenoisoquinolines LMP400 and LMP776) and three topoisomerase II inhibitors (etoposide, doxorubicin and ICRF-193). Highly significant correlations were found among Top1 inhibitors as well as Top2 inhibitors, while the profiles of Top1 inhibitors were different from those of Top2 inhibitors. Most distinct repair pathways between Top1 and Top2 inhibitors include NHEJ, TDP1, TDP2, PARP1 and Fanconi Anemia genes whereas HR appears relevant especially for Top1 and to a lesser extent for Top2 inhibitors. We also found and discuss differential pathways among Top1 inhibitors and Top2 inhibitors.

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Ubc13 is a ubiquitin-conjugating enzyme responsible for noncanonical ubiquitination of TNF receptor-associated factor (TRAF)-family adapter proteins involved in Toll-like receptor and TNFfamily cytokine receptor signaling, which are regulators of innate immunity. Gene ablation was used to study the function of Ubc13 in mice. Whereas homozygous ubc13 gene disruption resulted in embryonic lethality, heterozygous ubc13 ؉/؊ mice appeared normal, without alterations in immune cell populations. Haploinsufficient ubc13 ؉/؊ mice were resistant to lipopolysaccharide-induced lethality, and demonstrated reduced in vivo ubiquitination of TRAF6. Macrophages and splenocytes isolated from ubc13 ؉/؊ mice exhibited reduced lipopolysaccharide-inducible cytokine secretion and impaired activation of TRAF-dependent signal transduction pathways (NF-B, JNK, and p38 MAPK). These findings document a critical role for Ubc13 in inflammatory responses and suggest that agents reducing Ubc13 activity could have therapeutic utility.inflammation ͉ Toll-like receptor ͉ sepsis ͉ innate immunity