Murine cells homozygous for the severe combined immune deficiency mutation (scid) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair. Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PKcs, the catalytic subunit of this enzyme. Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PKcs gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PKcs is encoded by the XRCC7 gene. As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes.

Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.

The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cells, is defective in DNA double-strand break repair and in ability to undergo V(D)J recombination. The human XRCC5 DNA repair gene, which complements this mutant, is shown here through genetic and biochemical evidence to be the 80-kilodalton subunit of the Ku protein. Ku binds to free double-stranded DNA ends and is the DNA-binding component of the DNA-dependent protein kinase. Thus, the Ku protein is involved in DNA repair and in V(D)J recombination, and these results may also indicate a role for the Ku-DNA-dependent protein kinase complex in those same processes.

The human RAD51 protein is a homologue of the bacteria RecA and yeast RAD51 proteins that are involved in homologous recombination and DNA repair. RAD51 interacts with proteins involved in recombination and also with tumor suppressor proteins p53 and breast cancer susceptibility gene 1 (BRCA1). We have used the yeast two-hybrid system to clone murine cDNA sequences that encode two RAD51-associated molecules, RAB22 and RAB163. RAB163 encodes the C-terminal portion of mouse BRCA2, the homologue of the second breast cancer susceptibility gene protein in humans, demonstrating an in vitro association between RAD51 and BRCA2. RAB22 is a novel gene product that also interacts with RAD51 in vitro. To detect RAD51 interactions in vivo, we developed a transient nuclear focus assay that was used to demonstrate a complete colocalization of RAB22 with RAD51 in large nuclear foci.The RAD51 gene was originally identified in yeast as encoding a member of the RAD52 epistasis group (RAD50-RAD57) of proteins involved in DNA recombination and repair. RAD51 mutants are deficient in the repair of DNA damage caused by ionizing radiation, genetic recombination, and recombinational repair of the DNA lesions (1, 2). The RAD51 gene is well-conserved among eukaryotes (3), and the RAD51 protein is significantly homologous to the Escherichia coli RecA protein. Recent biochemical studies demonstrated that both yeast and human RAD51 proteins had the ability to promote ATP-dependent homologous pairing and strand-transfer reactions in vitro (2,4,5); but the precise function(s) of the mammalian RAD51 protein in vivo is not entirely clear. Targeted disruption of the mouse RAD51 gene resulted in an early embryonic lethal phenotype and also demonstrated that RAD51 is essential for cell proliferation (6, 7). Although targeted disruption can be a useful approach for understanding the overall function of genes in vivo, the early embryonic lethal phenotype of the RAD51 knock-out did not provide the information needed to elucidate specific functions of RAD51. Therefore, we have undertaken the strategy of cloning RAD51-associated molecules to investigate their functional role in the nuclear organization of RAD51.Yeast RAD51 belongs to the RAD52 epistasis group whose members are functionally associated. Yeast RAD52 and RAD55 directly associate with RAD51 (8), and human RAD52 also associates with human RAD51 (9). RecA activity is essential for the SOS response, a bacterial DNA damage control pathway that is dependent on gene transcription (10); notably, RAD51 is a member of the RNA polymerase II complex (10, 11). Defects in repair molecules such as RAD51 and associated molecules could perturb genomic integrity and eventually lead to tumorigenesis. Such a model has been proposed for mutations in mismatch repair molecules in hereditary nonpolyposis colon cancer (12). Recently, RAD51 was reported to interact with the breast cancer susceptibility gene 1 (BRCA1) duplicated (10) and the p53 tumor suppressor gene product (13). Although it is not yet clear ...

The gene product of XRCC4 has been implicated in both V(D)J recombination and the more general process of double strand break repair (DSBR). To date its role in these processes is unknown. Here, we describe biochemical characteristics of the murine XRCC4 protein. XRCC4 expressed in insect cells exists primarily as a disulfide-linked homodimer, although it can also form large multimers. Recombinant XRCC4 is phosphorylated during expression in insect cells. XRCC4 phosphorylation in Sf9 cells occurs on serine, threonine, and tyrosine residues.We also investigated whether XRCC4 interacts with the other factor known to be requisite for both V(D)J recombination and DSBR, the DNA-dependent protein kinase. We report that XRCC4 is an efficient in vitro substrate of DNA-PK and another unidentified serine/ threonine protein kinase(s). Both DNA-PK dependent and independent phosphorylation of XRCC4 in vitro occurs only on serine and threonine residues within the COOH-terminal 130 amino acids, a region of the molecule that is not absolutely required for XRCC4's DSBR function. Finally, recombinant XRCC4 facilitates Ku binding to DNA, promoting assembly of DNA-PK and complexing with DNA-PK bound to DNA. These data are consistent with the hypothesis that XRCC4 functions as an alignment factor in the DNA-PK complex.V(D)J recombination is the process of assembling the variable (V), diversity (D), and joining (J) gene segments of the immunoglobulin and T cell receptor variable region genes during development of B and T lymphocytes (1, 2). The proteins responsible for V(D)J joining are collectively referred to as the V(D)J recombinase, and are shared by both the B and T cell lineages. It has recently become clear that the lymphocytespecific proteins, recombination activating genes 1 and 2 (RAG 1 1 and RAG 2) (3, 4) are directly responsible for initiation of the V(D)J recombination reaction (5-8) together directing cleavage at the recombination signal sequence-coding juncture; whereas resolution of recombination intermediates also requires several ubiquitously expressed DNA repair factors. Evidence linking DNA repair activities with V(D)J recombination arose from the characterization of cells from homozygous severe combined immune deficient (SCID) mice (9 -11). C.B-17 SCID mice which are immunodeficient because of defective V(D)J recombination, were shown to also be defective in repairing DSBs in lymphoid and non-lymphoid cells (12, 13). Further evidence for an overlap in the activities of V(D)J recombination and DSB repair came from the observation that radiosensitive rodent cell lines which were defective in DSB repair could not support V(D)J recombination induced by co-transfecting RAG 1 and RAG 2; whereas, radiosensitive mutants proficient in DSB rejoining were normal in this capacity (14 -16). Four factors have been delineated which are required for both V(D)J recombination and DSBR; three of these encode components of the DNA-dependent protein kinase (17-25). DNA-PK is a nuclear serine/ threonine protein kinase whose catalytic ...

The catalyst system obtained by the equimolar reaction of diethylaluminium chloride with a,p,y,&tetraphenylporphine exhibits high catalytic activity in the polymerization of propylene oxide to give a polymer with a narrow molecular weight distribution. Average molecular weight of the polymer can be controlled by the initial mole ratio of propylene oxide to catalyst or by the conversion. Ethylene oxide also produces a polymer having a narrow molecular weight distribution.

Apoptosis and necrosis, two major forms of cell death, can be distinguished morphologically and biochemically. Internucleosomal DNA fragmentation (INDF) is a biochemical hallmark of apoptosis, and caspase-activated DNase (CAD), also known as DNA fragmentation factor 40 kDa (DFF40), is one of the major effector endonucleases. DNase γ, a Mg2+/Ca2+-dependent endonuclease, is also known to generate INDF but its role among other apoptosis-associated endonucleases in cell death is unclear. Here we show that (i) INDF occurs even during necrosis in cell lines, primary cells, and in tissues of mice in vivo, and (ii) DNase γ, but not CAD, is the effector endonuclease for INDF in cells undergoing necrosis. These results document a previously unappreciated role for INDF in necrosis and define its molecular basis.

XRCC4 is a generally expressed protein of 334 amino acids that is involved in the repair of DNA double-strand breaks and in V(D)J recombination, but its function is unknown. In this study, we have used a mutational approach and the yeast two-hybrid method to perform an initial characterization of this protein. We show that the XRCC4 protein is located in the nucleus. We also demonstrate that several potential phosphorylation sites are not required for XRCC4 function in a transient V(D)J recombination assay. In addition, we show that XRCC4 forms a homodimer in vivo with the homodimerization domain being located within amino acids 115-204. Finally, we define a core domain of XRCC4 that functions in V(D)J recombination and comprises amino acids 18-204. Potential functions of XRCC4 are discussed.