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NKG2D is a receptor expressed by NK cells and subsets of T lymphocytes. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We cloned and expressed two rat NKG2D ligands, both members of the RAE1 family, RAE1L and RRLT, and demonstrate that these ligands can induce IFN-gamma secretion and cytotoxicity by rat NK cells. To examine changes in expression of NKG2D and the NKG2D ligands RAE1L and RRLT after transplantation, we used a Dark Agouti (DA)-->Lewis rat model of liver transplantation. NKG2D expression was significantly increased in allogeneic liver grafts by day 7 post-transplant. Ligands of NKG2D, absent in normal liver, were readily detected in both syngeneic and allogeneic liver grafts by day 1 post-transplant. By day 7 post-transplant, hepatocyte RAE1L and RRLT expression was significantly and specifically increased in liver allografts. In contrast to acute rejection that develops in the DA-->Lewis model, transplantation of Lewis livers into DA recipients (Lewis-->DA) results in spontaneous tolerance. Interestingly, expression of RAE1L and RRLT is low in Lewis-->DA liver allografts, but significantly increased in DA-->Lewis liver allografts undergoing rejection. In conclusion, our results suggest that expression of NKG2D ligands may be important in allograft rejection.

International audience; Using proliferating cell nuclear antigen affinity chroma-tography and glycerol gradient centrifugation of partially purified fractions from mouse FM3A cells we have been able to isolate novel complexes of DNA polymerase delta and DNA ligase 1 containing clearly defined subunit compositions. In addition to the well known catalytic subunit of 125 kDa and accessory subunit of 48 kDa, the DNA polymerase delta complex contained three supplementary components, one of which reacted with antibodies directed against the p40 and p37 subunits of RF-C. Of the two remaining components, one termed p66 turned out to be coded by a gene whose putative C-terminal domain displayed significant homology with that of the Cdc27 subunit of Schizosaccharomyces pombe polymerase delta. On the basis of these and other observations, we propose p66 to be the missing third subunit of mammalian DNA polymerase delta. The DNA ligase 1 complex was made up of three novel components in addition to the 125 kDa catalytic subunit, two of which, p48 and p66, were common to DNA polymerase delta. We discuss the implications of our findings within the current framework of our understanding of DNA replication.

International audience; We have isolated a mutant in fission yeast, in which mitosis is uncoupled from completion of DNA replication when DNA synthesis is impaired by a thermosensitive mutation in the gene encoding the catalytic subunit of DNA polymerase delta. By functional complementation, we cloned the wild-type gene and identified it as the recently cloned checkpoint gene crb2+/rhp9+. This gene has been implicated in the DNA damage checkpoint and acts in the Chk1 pathway. Unlike the deleted strain dcrb2, cells bearing the crb2-1 allele were not affected in the DNA repair checkpoint after UV or MMS treatment at 30 degrees C, but were defective in this checkpoint function when treated with MMS at 37 degrees C. We analysed the involvement of Crb2 in the S/M checkpoint by blocking DNA replication with hydroxyurea, by using S phase cdc mutants, or by overexpression of the mutant PCNA L68S. Both crb2 mutants were unable to maintain the S/M checkpoint at 37 degrees C. Furthermore, the crb2+ gene was required, together with the cds1+ gene, for the S/M checkpoint at 30 degrees C. Finally, both the crb2 deletion and the crb2-1 allele induced a rapid death phenotype in the poldeltats3 background at both 30 degrees C and 37 degrees C. The rapid death phenotype was independent of the checkpoint functions.

International audience; We have isolated and characterized DNA polymerase delta (pol delta) from two thermosensitive Schizosaccharomyces pombe strains, poldeltats1 and poldeltats3, mutated in two different evolutionarily conserved domains of the catalytic subunit. At the restrictive temperature of 37 degreesC poldeltats1 and poldeltats3 mutant strains arrest growth in the S phase of the cell cycle. We show that at low levels of primer ends, in vitro stimulation by proliferating cell nuclear antigen (PCNA) of mutant enzymes is lower than stimulation of wild-type pol delta. Affinity for primer (3'-OH) ends and processivity of mutant enzymes do not appear different from wild-type pol delta. In contrast, Vmax values are lower than the wild-type value. The major in vitro defect appears to be decreased stimulation of mutant enzymes by PCNA, resulting in reduced velocity of DNA synthesis. In addition, ts1 pol delta is not stimulated by low PCNA concentration at 37 degreesC, although low concentrations stimulate activity at 25 degreesC, suggesting that this thermolability at low levels of primer ends could be its critical defect in vivo. Thus, both ts1 and ts3 pol delta mutations are located in regions of the catalytic subunit that seem necessary, directly or indirectly, for its efficient interaction with PCNA.

International audience; We have generated proliferating cell nuclear antigen (PCNA) mutants by low fidelity PCR and screened for lethal mutations by testing for lack of complementation of a Schizosaccharomyces pombe strain disrupted for the pcn1 + gene. We thus identified eight lethal mutants out of the 50 cDNAs tested. Six were truncated in their C-terminal region due to the introduction of a stop codon within their coding sequences. Two were full-length with a single point mutation at amino acid 68 or 69. The two latter mutants were overexpressed in insect cells via a recombinant baculovirus and were purified. They were unable to stimulate DNA polymerase delta DNA replication activity on a poly(dA).oligo(dT) template. Cross-linking experiments showed that this was due to their inability to form trimers. Since these two mutations are adjacent and not located in a domain of the protein putatively involved in inter-monomer interactions, our results show that the beta-sheet betaF1 to which they belong must play an essential role in maintaining the 3-dimensional structure of S.pombe PCNA.