Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination.
V(D)J recombination requires both lymphoid-specific and generally expressed enzymatic activities. All three known generally expressed activities involved in V(D)J recombination are also involved in DNA double-strand break repair (DSBR). Two of these are components of the DNA-dependent protein kinase (DNA-PK) and include Ku80 and DNA-PK catalytic subunit (DNA-PKcs); the third, XRCC4, is a protein of unknown function. The Ku70 protein is an additional component of DNA-PK; Ku70 forms a heterodimer with Ku80 to generate the DNA end-binding component of the enzyme. To test putative functions for Ku70, we have used gene-targeted mutation to generate a murine embryonic stem cell line which lacks Ku70 expression. We find that the Ku70(-/-) cells produce no detectable Ku70 and very little Ku80, suggesting a direct interrelationship between their levels. Correspondingly, these cells lack the nonspecific DNA end-binding activity associated with Ku. Significantly, the Ku70(-/-) embryonic stem cells have markedly increased sensitivity to gamma-irradiation relative to Ku70(+/-) or wild-type embryonic stem cells. Furthermore, the Ku70(-/-) cells lack the ability to effectively rejoin signal and coding ends liberated in transiently introduced V(D)J recombination substrates by enforced RAG-1 and RAG-2 expression. We conclude that the Ku70 gene product is involved in DSBR and V(D)J recombination and confirm that the Ku70 gene can be classified as a member of the x-ray cross-complementation group 6 (XRCC6). Potential differences between the Ku70(-/-) and Ku80(-/-) V(D)J recombination defects are discussed.
Altered fidelity of mitotic chromosome transmission in cell cycle mutants of S. cerevisiae.
Thirteen of 14 temperature-sensitive mutants deficient in successive steps of mitotic chromosome transmission (cdc2, 4, 5, 6, 7, 8, 9, 13, 14, 15, 16, 17 and 20) from spindle pole body separation to a late stage of nuclear division exhibited a dramatic increase in the frequency of chromosome loss and/or mitotic recombination when they were grown at their maximum permissive temperatures. The increase in chromosome loss and/or recombination is likely to be due to the deficiency of functional gene product rather than to an aberrant function of the mutant gene product since the mutant alleles are, with one exception, recessive to the wild-type allele for this phenotype. The generality of this result suggests that a delay in almost any stage of chromosome replication or segregation leads to a decrease in the fidelity of mitotic chromosome transmission. In contrast, temperature-sensitive mutants defective in the control step of the cell cycle (cdc28), in cytokinesis (cdc3) or in protein synthesis (ils1) did not exhibit increased recombination or chromosome loss.--Based upon previous results with mutants and DNA-damaging agents in a variety of organisms, we suggest that the induction of mitotic recombination in certain mutants is due to the action of a repair pathway upon nicks or gaps left in the DNA. This interpretation is supported by the fact that the induced recombination is dependent upon the RAD52 gene product, as essential component in the recombinogenic DNA repair pathway. Gene products whose deficiency leads to induced recombination are, therefore, strong candidates for proteins that function in DNA metabolism. Among the mutants that induce recombination are those known to be defective in some aspect of DNA replication (cdc2, 6, 8, 9) as well as some mutants defective in the G2 (cdc13 and 17) and M (cdc5 and 14) phases of the mitotic cycle. We suggest that special aspects of DNA metabolism may be occurring in G2 and M in order to prepare the chromosomes for proper segregation.
genetic algorithm positioning system process control sample size solar cell visible light dna sequence learning object indoor positioning received signal strength statistical process control indoor localization quantum dot statistical proces indoor positioning system count datum hecke algebra factorial design ieee standard binding site escherichia coli weighted moving average knowledge structure statistical quality control poisson structure cell cycle choice behavior econometric model quality level exponentially weighted moving fractional factorial design saccharomyces cerevisiae selection bia affine weyl group statistical process monitoring power conversion efficiency dye-sensitized solar cell charge transport uniform resource identifier learning object metadatum embryonic stem cell moving average control object class dye-sensitized solar reusable learning object linkage disequilibrium quantity discount spatial process spatial econometric population parameter embryonic stem reusable learning object metadatum heterojunction solar cell dna repair location fingerprinting cell development indoor positioning technique spatial econometric model radiation tolerance heterojunction solar genetic linkage signal peptide bulk heterojunction dna segment recombination rate bulk heterojunction solar dna recombination wifi-based indoor localization surface recombination escherichia coli. low-density lipoprotein indoor positioning solution proposed positioning system surface recombination velocity solar cells. neisseria meningitidi genetic heterogeneity learning object review dna break xrcc5 wt allele xrcc5 gene t cell receptor v(d)j recombination v(d)j recombination-activating protein 1 excretory function neuritis, autoimmune, experimental leukemia, b-cell dna sequence rearrangement immunoglobulin class switch recombination immunoglobulin class switching lipoprotein receptor dna breaks, double-stranded telomere maintenance v(d)j recombination genome encoded entity vdj recombinase recombination, genetic crossover (genetic algorithm) meiotic recombination homologous recombination