ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway

The rare diseases ataxia-telangiectasia (AT), caused by mutations in the ATM gene, and Nijmegen breakage syndrome (NBS), with mutations in the p95/nbs1 gene, share a variety of phenotypic abnormalities such as chromosomal instability, radiation sensitivity and defects in cell-cycle checkpoints in response to ionizing radiation. The ATM gene encodes a protein kinase that is activated by ionizing radiation or radiomimetic drugs, whereas p95/nbs1 is part of a protein complex that is involved in responses to DNA double-strand breaks. Here, because of the similarities between AT and NBS, we evaluated the functional interactions between ATM and p95/nbs1. Activation of the ATM kinase by ionizing radiation and induction of ATM-dependent responses in NBS cells indicated that p95/nbs1 may not be required for signalling to ATM after ionizing radiation. However, p95/nbs1 was phosphorylated on serine 343 in an ATM-dependent manner in vitro and in vivo after ionizing radiation. A p95/nbs1 construct mutated at the ATM phosphorylation site abrogated an S-phase checkpoint induced by ionizing radiation in normal cells and failed to compensate for this functional deficiency in NBS cells. These observations link ATM and p95/nbs1 in a common signalling pathway and provide an explanation for phenotypic similarities in these two diseases.     

Distribution of the 1,25 dihydroxy-vitamin D3 receptor in the bursa of Fabricius of chicken.

The vitamin D3 metabolite 1,25(OH)2D3 is probably involved in B lymphocyte ontogeny. We therefore determined the distribution of the 1,25(OH)2D3 receptor in the bursa of Fabricius and spleen cells of 7-day-old chicks, by immunohistochemistry using a monoclonal antibody against the chick intestinal cell 1,25(OH)2D3 receptor. The bursa cells of young (7-day-old) chicks contained large amounts of receptor while the spleen cells did not. The bursa cells of older (35-day-old) chicks contained fewer receptors, but the number of receptors in the spleen increased.     

Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres

Telomeres allow cells to distinguish natural chromosome ends from damaged DNA and protect the ends from degradation and fusion. In human cells, telomere protection depends on the TTAGGG repeat binding factor, TRF2 (refs 1-4), which has been proposed to remodel telomeres into large duplex loops (t-loops). Here we show by nanoelectrospray tandem mass spectrometry that RAD50 protein is present in TRF2 immunocomplexes. Protein blotting showed that a small fraction of RAD50, MRE11 and the third component of the MRE11 double-strand break (DSB) repair complex, the Nijmegen breakage syndrome protein (NBS1), is associated with TRF2. Indirect immunofluorescence demonstrated the presence of RAD50 and MRE11 at interphase telomeres. NBS1 was associated with TRF2 and telomeres in S phase, but not in G1 or G2. Although the MRE11 complex accumulated in irradiation-induced foci (IRIFs) in response to gamma-irradiation, TRF2 did not relocate to IRIFs and irradiation did not affect the association of TRF2 with the MRE11 complex, arguing against a role for TRF2 in DSB repair. Instead, we propose that the MRE11 complex functions at telomeres, possibly by modulating t-loop formation.     

Expression of multidrug resistance (mdr) gene(s) in primary lymphoid organs of chicken immune system during embryonic development

The presence of a multidrug resistance (MDR) related protein, P-170, in normal and pathological lymphoid cells has been described. The present report evaluates the expression of themdr 1 gene by using the reverse Polymerase Chain Reaction (PCR) on cells obtained from the thymus and bursa of chicken embryos starting from day 12 until hatching. Results show that the thymic cells are positive from day 12 to the end of the observation period. In contrast,mdr mRNA was detected in the bursa from day 14 to day 17 of embryonic life. Possible relationships between the expression ofmdr and the development of T and B lymphocytes are discussed.     

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 A 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 Zn(2+) 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 A. 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.     

Distribution of the 1,25 dihydroxy-vitamin D3 receptor in the bursa of Fabricius of chicken

The vitamin D₃ metabolite 1,25(OH)₂D₃ is probably involved in B lymphocyte ontogeny. We therefore determined the distribution of the 1,25(OH)₂D₃ receptor in the bursa of Fabricius and spleen cells of 7-day-old chicks, by immunohistochemistry using a monoclonal antibody against the chick intestinal cell 1,25(OH)₂D₃ receptor. The bursa cells of young (7-day-old) chicks contained large amounts of receptor while the spleen cells did not. The bursa cells of older (35-day-old) chicks contained fewer receptors, but the number of receptors in the spleen increased.     

The DNA damage-dependent intra-S phase checkpoint is regulated by parallel pathways

To preserve genetic integrity, mammalian cells exposed to ionizing radiation activate the ATM kinase, which initiates a complex response-including the S-phase checkpoint pathways-to delay DNA replication. Defects in ATM or its substrates Nbs1 or Chk2 (ref. 3), the Nbs1-interacting Mre11 protein, or the Chk2-regulated Cdc25A-Cdk2 cascade all cause radio-resistant DNA synthesis (RDS). It is unknown, however, whether these proteins operate in a common signaling cascade. Here we show that experimental blockade of either the Nbs1-Mre11 function or the Chk2-triggered events leads to a partial RDS phenotype in human cells. In contrast, concomitant interference with Nbs1-Mre11 and the Chk2-Cdc25A-Cdk2 pathways entirely abolishes inhibition of DNA synthesis induced by ionizing radiation, resulting in complete RDS analogous to that caused by defective ATM. In addition, Cdk2-dependent loading of Cdc45 onto replication origins, a prerequisite for recruitment of DNA polymerase, was prevented upon irradiation of normal or Nbs1/Mre11-defective cells but not cells with defective ATM. We conclude that in response to ionizing radiation, phosphorylations of Nbs1 and Chk2 by ATM trigger two parallel branches of the DNA damage-dependent S-phase checkpoint that cooperate by inhibiting distinct steps of DNA replication.     

An alternative mode of translation permits production of a variant NBS1 protein from the common Nijmegen breakage syndrome allele

Nijmegen breakage syndrome (NBS) is a rare chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity and radioresistant DNA synthesis-S phase checkpoint deficiency, which results in the failure to suppress DNA replication origins following DNA damage. Approximately 90% of NBS patients are homozygous for the 657del5 allele, a truncating mutation of NBS1 that causes premature termination at codon 219. Because null mutations in MRE11 and RAD50, which encode binding partners of NBS1, are lethal in vertebrates, and mouse Nbs1-null mutants are inviable, we tested the hypothesis that the NBS1 657del5 mutation was a hypomorphic defect. We showed that NBS cells contain the predicted 26-kD amino-terminal protein fragment, NBS1p26, and a 70-kD NBS1 protein (NBS1p70) lacking the native N terminus. The NBSp26 protein is not physically associated with the MRE11 complex, whereas the p70 species is physically associated with it. NBS1p70 is produced by internal translation initiation within the NBS1 mRNA using an open reading frame generated by the 657del5 frameshift. We propose that the common NBS1 allele encodes a partially functional protein that diminishes the severity of the NBS phenotype.     

The BRCA1-interacting helicase BRIP1 is deficient in Fanconi anemia

Seven Fanconi anemia-associated proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG and FANCL) form a nuclear Fanconi anemia core complex that activates the monoubiquitination of FANCD2, targeting FANCD2 to BRCA1-containing nuclear foci. Cells from individuals with Fanconi anemia of complementation groups D1 and J (FA-D1 and FA-J) have normal FANCD2 ubiquitination. Using genetic mapping, mutation identification and western-blot data, we identify the defective protein in FA-J cells as BRIP1 (also called BACH1), a DNA helicase that is a binding partner of the breast cancer tumor suppressor BRCA1.