The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair
The Mre11 complex (Mre11–Rad50–Nbs1) is central to chromosomal maintenance and functions in homologous recombination, telomere maintenance and sister chromatid association. These functions all imply that the linked binding of two DNA substrates occurs, although the molecular basis for this process remains unknown. Here we present a 2.2 Å crystal structure of the Rad50 coiled-coil region that reveals an unexpected dimer interface at the apex of the coiled coils in which pairs of conserved Cys-X-X-Cys motifs form interlocking hooks that bind one Zn2+ ion. Biochemical, X-ray and electron microscopy data indicate that these hooks can join oppositely protruding Rad50 coiled-coil domains to form a flexible bridge of up to 1,200 Å. This suggests a function for the long insertion in the Rad50 ABC-ATPase domain. The Rad50 hook is functional, because mutations in this motif confer radiation sensitivity in yeast and disrupt binding at the distant Mre11 nuclease interface. These data support an architectural role for the Rad50 coiled coils in forming metal-mediated bridging complexes between two DNA-binding heads. The resulting assemblies have appropriate lengths and conformational properties to link sister chromatids in homologous recombination and DNA ends in non-homologous end-joining.
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.
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