DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-stranded break repair and V(D)J recombination, comprises a DNA-targeting component called Ku and an approximately 460 kDa catalytic subunit, DNA-PKcs. Here, we describe the cloning of the DNA-PKcs cDNA and show that DNA-PKcs falls into the phosphatidylinositol (PI) 3-kinase family. Biochemical assays, however, indicate that DNA-PK phosphorylates proteins but has no detectable activity toward lipids. Strikingly, DNA-PKcs is most similar to PI kinase family members involved in cell cycle control, DNA repair, and DNA damage responses. These include the FKBP12-rapamycin-binding proteins Tor1p, Tor2p, and FRAP, S. pombe rad3, and the product of the ataxia telangiectasia gene, mutations in which lead to genomic instability and predisposition to cancer. The relationship of these proteins to DNA-PKcs provides important clues to their mechanisms of action.

DNA-dependent protein kinase (DNA-PK) consists of a heterodimeric protein (Ku) and a large catalytic subunit (DNA-PKcs). The Ku protein has double-stranded DNA end-binding activity that serves to recruit the complex to DNA ends. Despite having serine/threonine protein kinase activity, DNA-PKcs falls into the phosphatidylinositol 3-kinase superfamily. DNA-PK functions in DNA double-strand break repair and V(D)J recombination, and recent evidence has shown that mouse scid cells are defective in DNA-PKcs. In this study we have cloned the cDNA for the carboxyl-terminal region of DNA-PKcs in rodent cells and identifed the existence of two differently spliced products in human cells. We show that DNA-PKcs maps to the same chromosomal region as the mouse scid gene. scid cells contain approximately wild-type levels of DNA-PKcs transcripts, whereas the V-3 cell line, which is also defective in DNA-PKcs, contains very reduced transcript levels. Sequence comparison of the carboxylterminal region of scid and wild-type mouse cells enabled us to identify a nonsense mutation within a highly conserved region of the gene in mouse scid cells. This represents a strong candidate for the inactivating mutation in DNA-PKcs in the scid mouse.In 1983 Bosma et al.(1) observed a severe combined immunodeficient (scid) mouse in a litter of otherwise normal mice and subsequent backcrossing established the now well-known scid mouse. Only now are details of the molecular defect in scid mice beginning to emerge. scid mice and cell lines derived from them have an interesting and pleiotropic phenotype, with features certainly unpredicted in 1983 (2). scid mice fail to develop mature T and B lymphocytes due to an inability to carry out functional rearrangements of the elements encoding the immunoglobulin and T-cell receptor genes (3-6). These elements, termed the variable (V), diversity (D), and joining (J) segments, are spatially separated in germ-line cells but are rearranged into a contiguous unit during maturation of T and B cells in the process called V(D)J rearrangement (for reviews, see refs. 7-9). This process is initiated by double-strand breaks (dsbs) introduced between partially conserved recombination signal sequences (RSS) and the flanking sequence encoding the V, D, or J element (coding sequence). The rearrangement yields one junction in which the two RSS sequences are rejoined precisely and a second in which the two coding elements are rejoined. The latter junctions invariably harbor small deletions and insertions. scid cells manifest a SCID phenotype due to an inability to form functional coding junctions. In contrast, signal junctions are formed at near normal levels (3-6).In lower organisms, many mutants defective in processes of genetic recombination are sensitive to ionizing radiation (10). This link prompted an examination of scid cell lines, which proved to be both radiosensitive and defective in their ability to rejoin DNA dsbs (11)(12)(13). A number of radiosensitive dsb-repair-defective hamster cell mutants have al...

The DNA-dependent protein kinase is a mammalian protein complex composed of Ku70, Ku80, and DNA-PKcs subunits that has been implicated in DNA double-strand break repair and V(D)J recombination. Here, by gene targeting, we have constructed a mouse with a disruption in the kinase domain of DNA-PKcs, generating an animal model completely devoid of DNA-PK activity. Our results demonstrate that DNA-PK activity is required for coding but not for signal join formation in mice. Although our DNA-PKcs defective mice closely resemble Scid mice, they differ by having elevated numbers of CD4+CD8+ thymocytes. This suggests that the Scid mice may not represent a null phenotype and may retain some residual DNA-PKcs function.

In mammalian cells, the Ku and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are required for the correct and efficient repair of DNA double-strand breaks. Ku comprises two tightly-associated subunits of approximately 69 and approximately 83 kDa, which are termed Ku70 and Ku80 (or Ku86), respectively. Previously, a number of regions of both Ku subunits have been demonstrated to be involved in their interaction, but the molecular mechanism of this interaction remains unknown. We have identified a region in Ku70 (amino acid residues 449-578) and a region in Ku80 (residues 439-592) that participate in Ku subunit interaction. Sequence analysis reveals that these interaction regions share sequence homology and suggests that the Ku subunits are structurally related. On binding to a DNA double-strand break, Ku is able to interact with DNA-PKcs, but how this interaction is mediated has not been defined. We show that the extreme C-terminus of Ku80, specifically the final 12 amino acid residues, mediates a highly specific interaction with DNA-PKcs. Strikingly, these residues appear to be conserved only in Ku80 sequences from vertebrate organisms. These data suggest that Ku has evolved to become part of the DNA-PK holo-enzyme by acquisition of a protein-protein interaction motif at the C-terminus of Ku80.

Diseases affecting hemoglobin synthesis and function are extremely common worldwide. More than 1000 naturally occurring human hemoglobin variants with single amino acid substitutions throughout the molecule have been discovered, mainly through their clinical and/or laboratory manifestations. These variants alter hemoglobin structure and biochemical properties with physiological effects ranging from insignificant to severe. Studies of these mutations in patients and in the laboratory have produced a wealth of information on hemoglobin biochemistry and biology with significant implications for hematology practice. More generally, landmark studies of hemoglobin performed over the past 60 years have established important paradigms for the disciplines of structural biology, genetics, biochemistry, and medicine. Here we review the major classes of hemoglobin variants, emphasizing general concepts and illustrative examples.

Hemoglobin A (HbA), the oxygen delivery system in humans, comprises two alpha and two beta subunits. Free alpha-hemoglobin (alphaHb) is unstable, and its precipitation contributes to the pathophysiology of beta thalassemia. In erythrocytes, the alpha-hemoglobin stabilizing protein (AHSP) binds alphaHb and inhibits its precipitation. The crystal structure of AHSP bound to Fe(II)-alphaHb reveals that AHSP specifically recognizes the G and H helices of alphaHb through a hydrophobic interface that largely recapitulates the alpha1-beta1 interface of hemoglobin. The AHSP-alphaHb interactions are extensive but suboptimal, explaining why beta-hemoglobin can competitively displace AHSP to form HbA. Remarkably, the Fe(II)-heme group in AHSP bound alphaHb is coordinated by the distal but not the proximal histidine. Importantly, binding to AHSP facilitates the conversion of oxy-alphaHb to a deoxygenated, oxidized [Fe(III)], nonreactive form in which all six coordinate positions are occupied. These observations reveal the molecular mechanisms by which AHSP stabilizes free alphaHb.

The transactivation/transformation-domain associated protein (TRRAP) belongs to the Ataxia-telangiectasia mutated (ATM) super-family and has been identified as a cofactor for c-MYC-mediated oncogenic transformation. TRRAP and its yeast homolog (Tra1p) are components of histone acetyltransferase (HAT) complexes, SAGA (refs. 2,4,5), PCAF (ref. 3) and NuA4 (ref. 6), which are important for the regulation of transcription and cell cycle progression and also have a role in cell viability. Yet the biological function of this molecule and how it controls proliferation are still unclear. Here we show that null mutation of Trrap in mice results in peri-implantation lethality due to a blocked proliferation of blastocysts. We use an inducible Cre-loxP system to show that loss of Trrap blocks cell proliferation because of aberrant mitotic exit accompanied by cytokinesis failure and endoreduplication. Trrap-deficient cells fail to sustain mitotic arrest despite chromosome missegregation and disrupted spindles, and display compromised cdk1 activity. Trrap is therefore essential for early development and required for the mitotic checkpoint and normal cell cycle progression.

The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensively studied xrs mutants, has recently been identified as Ku80, a subunit of the Ku protein and a component of DNA-dependent protein kinase (DNA-PK). Several group 5 mutants, including xrs-5 and -6, lack double-stranded DNA end-binding and DNA-PK activities. In this study, we examined additional xrs mutants at the molecular and biochemical levels. All mutants examined have low or undetectable levels of Ku70 and Ku80 protein, end-binding, and DNA-PK activities. Only one mutant, xrs-6, has Ku80 transcript levels detectable by Northern hybridization, but Ku80 mRNA was detectable by reverse transcription-PCR in most other mutants. Two mutants, xrs-4 and -6, have altered Ku80 transcripts resulting from mutational changes in the genomic Ku80 sequence affecting RNA splicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression. Neither of these two mutants has detectable wild-type Ku80 transcript. Since the mutation in both xrs-4 and xrs-6 cells results in severely truncated Ku80 protein, both are likely candidates to be null mutants. Azacytidine-induced revertants of xrs-4 and -6 carried both wild-type and mutant transcripts. The results with these revertants strongly support our model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation. Site-directed mutagenesis studies indicate that previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities.