scholarly journals Ku86 defines the genetic defect and restores X-ray resistance and V(D)J recombination to complementation group 5 hamster cell mutants.

1996 ◽  
Vol 16 (4) ◽  
pp. 1519-1526 ◽  
Author(s):  
A Errami ◽  
V Smider ◽  
W K Rathmell ◽  
D M He ◽  
E A Hendrickson ◽  
...  
Keyword(s):  
Mutant Cell ◽  
Genetic Defect ◽  
Strand Break ◽  
Complementation Group ◽  
X Rays ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Gene Encoding ◽  
X Ray ◽  
Group 5

X-ray-sensitive hamster cells in complementation groups 4, 5, 6, and 7 are impaired for both double-strand break repair and V(D)J recombination. Here we show that in two mutant cell lines (XR-V15B and XR-V9B) from group 5, the genetic defects are in the gene encoding the 86-kDa subunit of the Ku autoantigen, a nuclear protein that binds to the double-stranded DNA ends. These mutants express Ku86 mRNA containing deletions of 138 and 252 bp, respectively, and the encoded proteins contain internal, in-frame deletions of 46 and 84 amino acids. Two X-ray-resistant revertants of XR-V15B expressed two Ku86 transcripts, one with and one without the deletion, suggesting that reversion occurred by activation of a silent wild-type allele. Transfection of full-length cDNA encoding hamster Ku86 into XR-V15B cells resulted in a complete rescue of DNA-end-binding (DEB) activity and Ku70 levels, suggesting that Ku86 stabilizes the Ku70 polypeptide. In addition, cells expressing wild-type levels of DEB activity were fully rescued for X-ray resistance and V(D)J recombination, whereas cells expressing lower levels of DEB activity were only partially rescued. Thus, Ku is an essential component of the pathway(s) utilized for the resolution of DNA double-strand breaks induced by either X rays or V(D)J recombination, and mutations in the Ku86 gene are responsible for the phenotype of group 5 cells.

scholarly journals MPH1, A Yeast Gene Encoding a DEAH Protein, Plays a Role in Protection of the Genome From Spontaneous and Chemically Induced Damage

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1069-1081 ◽  
Author(s):  
Jürgen Scheller ◽  
Anke Schürer ◽  
Christian Rudolph ◽  
Stefan Hettwer ◽  
Wilfried Kramer
Keyword(s):  
Uv Light ◽  
Missense Mutations ◽  
X Rays ◽  
Wild Type ◽  
Yeast Gene ◽  
Gene Encoding ◽  
Epistatic Interactions ◽  
Mutator Phenotype ◽  
X Ray ◽  
Chemically Induced

Abstract We have characterized the MPH1 gene from Saccharomyces cerevisiae. mph1 mutants display a spontaneous mutator phenotype. Homologs were found in archaea and in the EST libraries of Drosophila, mouse, and man. Mph1 carries the signature motifs of the DEAH family of helicases. Selected motifs were shown to be necessary for MPH1 function by introducing missense mutations. Possible indirect effects on translation and splicing were excluded by demonstrating nuclear localization of the protein and splicing proficiency of the mutant. A mutation spectrum did not show any conspicuous deviations from wild type except for an underrepresentation of frameshift mutations. The mutator phenotype was dependent on REV3 and RAD6. The mutant was sensitive to MMS, EMS, 4-NQO, and camptothecin, but not to UV light and X rays. Epistasis analyses were carried out with representative mutants from various repair pathways (msh6, mag1, apn1, rad14, rad52, rad6, mms2, and rev3). No epistatic interactions were found, either for the spontaneous mutator phenotype or for MMS, EMS, and 4-NQO sensitivity. mph1 slightly increased the UV sensitivity of mms2, rad6, and rad14 mutants, but no effect on X-ray sensitivity was observed. These data suggest that MPH1 is not part of a hitherto known repair pathway. Possible functions are discussed.

scholarly journals EVALUATION THE ROLE OF VITAMIN C AS A RADIATION PROTECTIVE AGENT USING γ-H2AX FOR SIGNALING OF DNA DAMAGE ON IRRADIATED MICE TESTIS

2020 ◽  
Vol 17 (36) ◽  
pp. 128-139
Author(s):  
Ekhlas A ALANI ◽  
Mustafa S ALMUSAWI ◽  
Amar H MAHDI
Keyword(s):  
Vitamin C ◽  
Control Group ◽  
Protective Agent ◽  
X Rays ◽  
Dna Double Strand Breaks ◽  
X Ray ◽  
Radiation Group ◽  
Significant Difference ◽  
Group 5 ◽  
Group 2

An interesting feature of ionizing radiation, especially Gamma and X-rays as a DNA damaging factor is the range of lesions it induces. γ-H2AX foci are documented to represent DNA double-strand breaks (DSBs) as a biomarker for radiation-induced damage. Study design 42 adult male mice Albino BALB/c, had been divided randomly into 6 groups of seven mice each. Group 1 received a standard saline solution untreated also, do not expose to radiation. Group 2 mice received vitamin C (VC) (200 mg/kg.day) intra-peritoneal (i.p.) injected for 8 days without radiation. Group 3 control was exposed to γ-radiation. Group 4 control was exposed to X-ray radiation. Group 5 mice had been administrated with vitamin C in the identical dose of group 2 for 8 days, then exposed to (4 Gy) of γ-ray. Group 6 was administrated with vitamin dose in the same above and the same period, then exposed to (4 Gy) of X-ray. All groups had been sacrificed by cervical dislocation at (1, 3, and 24 h). Post radiation testis mice tissues were collected. A significant difference (P 0.05) between the group of vitamin C and with a control group exposed to both (γ, X-rays) in foci forming, but there is no significant difference (P 0.05) between γ and X- rays for the control and vitamin C groups. The results demonstrate that vitamin C is a good radioprotective agent for testis mice tissues; the effect of (γ and X-rays) had almost the same results on the mice testicle tissues with the same dose.

scholarly journals A DNA end-binding factor involved in double-strand break repair and V(D)J recombination.

1994 ◽  
Vol 14 (7) ◽  
pp. 4741-4748 ◽  
Author(s):  
W K Rathmell ◽  
G Chu
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
X Rays ◽  
X Ray ◽  
Break Repair ◽  
Group 5 ◽  
Dna Ends

We have identified a nuclear factor that binds to double-stranded DNA ends, independently of the structure of the ends. It had equivalent affinities for DNA ends created by sonication or by restriction enzymes leaving 5', 3', or blunt ends but had no detectable affinity for single-stranded DNA ends. Since X rays induce DNA double-strand breaks, extracts from several complementation groups of X-ray-sensitive mammalian cells were tested for this DNA end-binding (DEB) activity. DEB activity was deficient in three independently derived cell lines from complementation group 5. Furthermore, when the cell lines reverted to X-ray resistance, expression of the DEB factor was restored to normal levels. Previous studies had shown that group 5 cells are defective for both double-strand break repair and V(D)J recombination. The residual V(D)J recombination activity in these cells produces abnormally large deletions at the sites of DNA joining (F. Pergola, M. Z. Zdzienicka, and M. R. Lieber, Mol. Cell. Biol. 13:3464-3471, 1993, and G. Taccioli, G. Rathbun, E. Oltz, T. Stamato, P. Jeggo, and F. Alt, Science 260:207-210, 1993), consistent with deficiency of a factor that protects DNA ends from degradation. Therefore, DEB factor may be involved in a biochemical pathway common to both double-strand break repair and V(D)J recombination.

A DNA end-binding factor involved in double-strand break repair and V(D)J recombination

1994 ◽  
Vol 14 (7) ◽  
pp. 4741-4748
Author(s):  
W K Rathmell ◽  
G Chu
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
X Rays ◽  
X Ray ◽  
Break Repair ◽  
Group 5 ◽  
Dna Ends

We have identified a nuclear factor that binds to double-stranded DNA ends, independently of the structure of the ends. It had equivalent affinities for DNA ends created by sonication or by restriction enzymes leaving 5', 3', or blunt ends but had no detectable affinity for single-stranded DNA ends. Since X rays induce DNA double-strand breaks, extracts from several complementation groups of X-ray-sensitive mammalian cells were tested for this DNA end-binding (DEB) activity. DEB activity was deficient in three independently derived cell lines from complementation group 5. Furthermore, when the cell lines reverted to X-ray resistance, expression of the DEB factor was restored to normal levels. Previous studies had shown that group 5 cells are defective for both double-strand break repair and V(D)J recombination. The residual V(D)J recombination activity in these cells produces abnormally large deletions at the sites of DNA joining (F. Pergola, M. Z. Zdzienicka, and M. R. Lieber, Mol. Cell. Biol. 13:3464-3471, 1993, and G. Taccioli, G. Rathbun, E. Oltz, T. Stamato, P. Jeggo, and F. Alt, Science 260:207-210, 1993), consistent with deficiency of a factor that protects DNA ends from degradation. Therefore, DEB factor may be involved in a biochemical pathway common to both double-strand break repair and V(D)J recombination.

scholarly journals The Internal Phosphodiesterase RegA Is Essential for the Suppression of Lateral Pseudopods duringDictyosteliumChemotaxis

2000 ◽  
Vol 11 (8) ◽  
pp. 2803-2820 ◽  
Author(s):  
Deborah J. Wessels ◽  
Hui Zhang ◽  
Joshua Reynolds ◽  
Karla Daniels ◽  
Paul Heid ◽  
...  
Keyword(s):  
Mutant Cell ◽  
Computer Assisted ◽  
Spatial Gradient ◽  
Wild Type ◽  
Gene Encoding ◽  
Camp Phosphodiesterase ◽  
Temporal Gradient ◽  
Spatial Gradients ◽  
Normal Manner ◽  
Analysis System

Dictyostelium strains in which the gene encoding the cytoplasmic cAMP phosphodiesterase RegA is inactivated form small aggregates. This defect was corrected by introducing copies of the wild-type regA gene, indicating that the defect was solely the consequence of the loss of the phosphodiesterase. Using a computer-assisted motion analysis system,regA−mutant cells were found to show little sense of direction during aggregation. When labeled wild-type cells were followed in a field of aggregatingregA−cells, they also failed to move in an orderly direction, indicating that signaling was impaired in mutant cell cultures. However, when labeled regA−cells were followed in a field of aggregating wild-type cells, they again failed to move in an orderly manner, primarily in the deduced fronts of waves, indicating that the chemotactic response was also impaired. Since wild-type cells must assess both the increasing spatial gradient and the increasing temporal gradient of cAMP in the front of a natural wave, the behavior of regA−cells was motion analyzed first in simulated temporal waves in the absence of spatial gradients and then was analyzed in spatial gradients in the absence of temporal waves. Our results demonstrate that RegA is involved neither in assessing the direction of a spatial gradient of cAMP nor in distinguishing between increasing and decreasing temporal gradients of cAMP. However, RegA is essential for specifically suppressing lateral pseudopod formation during the response to an increasing temporal gradient of cAMP, a necessary component of natural chemotaxis. We discuss the possibility that RegA functions in a network that regulates myosin phosphorylation by controlling internal cAMP levels, and, in support of that hypothesis, we demonstrate that myosin II does not localize in a normal manner to the cortex ofregA−cells in an increasing temporal gradient of cAMP.

The Effects of Heavy Ion Particles on Human Megakaryocytopoiesis and Thrombopoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4156-4156
Author(s):  
Ikuo Kashiwakura ◽  
Kenji Takahashi ◽  
Satoru Monzen ◽  
Kiyomi Eguchi-Kasai ◽  
Tsutomu Toki ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ion Beam ◽  
Heavy Ion ◽  
X Rays ◽  
Dna Double Strand Breaks ◽  
Nuclear Facilities ◽  
Plasma Clot ◽  
Carbon Ion ◽  
X Ray ◽  
Carbon Ion Beam

Abstract Heavy ion particles provide unique properties in radiotherapy. However, they have also been shown to pose high risks for both work at nuclear facilities and astronauts participating in space missions. In a previous study, we demonstrated that in radio-sensitive megakaryocyte progenitor cells, namely colony-forming unit megakaryocytes (CFU-Meg), a degree of X-ray-induced damage was prevented by post-treatment with several cytokines. In this study, we analyzed the effects of heavy ion particles on megakaryocytopoiesis and thrombopoiesis. The CD34+ CFU-Meg were isolated from human placental and umbilical cord blood using a magnetic isolation kit and then were exposed to a carbon ion beam (LET=50 KeV/mm). They were cultured in a serum free medium supplemented with a thrombopoietin (TPO) alone or a combination of TPO plus other cytokines including stem cell factor, interleukin-3 (IL-3) and Flt3-ligand. The number of CFU-Meg was calculated by a plasma clot technique. The differentiation into megakaryocytes (CD41+) and the release of platelets (CD42a+) in a liquid culture were both analyzed by flow cytometry. The increase of gamma-H2AX, a marker of DNA double-strand breaks (DSBs) was also detected by flow cytometry. The sensitivity of CFU-Meg to a carbon ion beam was found to be extremely high and could not be lowered by any type of cytokines unlike X-rays. However, treatment with TPO plus IL-3 potentially induced megakaryocytopoiesis and thrombopoiesis at 14 days after the exposure to a carbon ion beam at 2 Gy. The cytokine treatment enhanced the induction of gamma-H2AX in X-ray-irradiated CD34+ CFU-Meg but not in a carbon ion beam-irradiated one. These results show that not only the downregulation of death signals, but also the repair of DSBs was less strongly promoted by cytokines in CFU-Meg exposed to a carbon ion beam than X-rays. Different treatments therefore are required to protect against megakaryocytopoiesis and thrombopoiesis damage by heavy ion particles.

scholarly journals LigD: A Structural Guide to the Multi-Tool of Bacterial Non-Homologous End Joining

2021 ◽  
Vol 8 ◽  
Author(s):  
Benhur Amare ◽  
Anthea Mo ◽  
Noorisah Khan ◽  
Dana J. Sowa ◽  
Monica M. Warner ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Strand Break ◽  
Double Strand Break ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Repair Mechanism ◽  
End Joining ◽  
Dna Double Strand Break ◽  
Non Homologous End Joining ◽  
The Individual

DNA double-strand breaks are the most lethal form of damage for living organisms. The non-homologous end joining (NHEJ) pathway can repair these breaks without the use of a DNA template, making it a critical repair mechanism when DNA is not replicating, but also a threat to genome integrity. NHEJ requires proteins to anchor the DNA double-strand break, recruit additional repair proteins, and then depending on the damage at the DNA ends, fill in nucleotide gaps or add or remove phosphate groups before final ligation. In eukaryotes, NHEJ uses a multitude of proteins to carry out processing and ligation of the DNA double-strand break. Bacterial NHEJ, though, accomplishes repair primarily with only two proteins–Ku and LigD. While Ku binds the initial break and recruits LigD, it is LigD that is the primary DNA end processing machinery. Up to three enzymatic domains reside within LigD, dependent on the bacterial species. These domains are a polymerase domain, to fill in nucleotide gaps with a preference for ribonucleotide addition; a phosphoesterase domain, to generate a 3′-hydroxyl DNA end; and the ligase domain, to seal the phosphodiester backbone. To date, there are no experimental structures of wild-type LigD, but there are x-ray and nuclear magnetic resonance structures of the individual enzymatic domains from different bacteria and archaea, along with structural predictions of wild-type LigD via AlphaFold. In this review, we will examine the structures of the independent domains of LigD from different bacterial species and the contributions these structures have made to understanding the NHEJ repair mechanism. We will then examine how the experimental structures of the individual LigD enzymatic domains combine with structural predictions of LigD from different bacterial species and postulate how LigD coordinates multiple enzymatic activities to carry out DNA double-strand break repair in bacteria.

ATM and RAD51 Repair Pathways in Human Lymphocytes Irradiated with 70 MeV Therapeutic Proton Beam

2021 ◽  
Author(s):  
Agnieszka Panek ◽  
Justyna Miszczyk
Keyword(s):  
Proton Beam ◽  
Human Lymphocytes ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
X Rays ◽  
X Ray ◽  
Post Irradiation ◽  
Repair Protein ◽  
Radiation Induced

The repair of radiation-induced DNA damage is a key factor differentiating patients in terms of the therapeutic efficacy and toxicity to surrounding normal tissue. Proton energy substantially determines the types of cancers that can be treated. The present work investigated the DNA double-strand break repair systems, represented by phosphorylated ATM and Rad51. The status of proton therapy energy used to treat major types of cancer is summarized. Here, human lymphocytes from eight healthy donors (male and female) were irradiated with a spread-out Bragg peak using a therapeutic 70 MeV proton beam or with reference X rays. For both types of radiation, the kinetics of pATM and Rad51 repair protein activation (0–24 h) were estimated as determinants of homologous and non-homologous double-strand break repair. Additionally, γ-H2AX was used as the gold standard marker of double-strand breaks. Our results showed that at 30 min postirradiation there was significantly greater accumulation of γ-H2AX (0.6-fold), pATM (2.0-fold), and Rad51 (0.6-fold) in the proton-irradiated cells compared with the X-ray-treated cells. At 24 h post irradiation, for both types of radiation and all investigated proteins, the foci number was still significantly higher when compared with control. Furthermore, the mean value of pATM and Rad51 repair effectiveness was higher in cells exposed to protons than in cells exposed to X rays; however, the difference was significant only for pATM. The largest inter-individual differences in the repair capabilities were noted for Rad51. The association between the frequency of repair protein foci and the frequency of lymphocyte viability at 1 h post irradiation showed a positive correlation for protons but a negative correlation for X rays. These findings indicate that the accumulation of radiation-induced repair protein foci after proton versus X-ray irradiation differs between patients, consequently affecting the cellular responses to particle therapy and conventional radiation therapy.

scholarly journals DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions

2018 ◽  
Vol 115 (4) ◽  
pp. 762-767 ◽  
Author(s):  
Rohit A. Panchakshari ◽  
Xuefei Zhang ◽  
Vipul Kumar ◽  
Zhou Du ◽  
Pei-Chi Wei ◽  
...  
Keyword(s):  
Chromosomal Translocation ◽  
Strand Break ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Response Factors ◽  
Class Switch ◽  
B Lymphoma Cells ◽  
Donor And Acceptor ◽  
Primary Mouse

Ig heavy chain (IgH) class switch recombination (CSR) in B lymphocytes switches IgH constant regions to change antibody functions. CSR is initiated by DNA double-strand breaks (DSBs) within a donor IgH switch (S) region and a downstream acceptor S region. CSR is completed by fusing donor and acceptor S region DSB ends by classical nonhomologous end-joining (C-NHEJ) and, in its absence, by alternative end-joining that is more biased to use longer junctional microhomologies (MHs). Deficiency for DSB response (DSBR) factors, including ataxia telangiectasia-mutated (ATM) and 53BP1, variably impair CSR end-joining, with 53BP1 deficiency having the greatest impact. However, studies of potential impact of DSBR factor deficiencies on MH-mediated CSR end-joining have been technically limited. We now use a robust DSB joining assay to elucidate impacts of deficiencies for DSBR factors on CSR and chromosomal translocation junctions in primary mouse B cells and CH12F3 B-lymphoma cells. Compared with wild-type, CSR and c-myc to S region translocation junctions in the absence of 53BP1, and, to a lesser extent, other DSBR factors, have increased MH utilization; indeed, 53BP1-deficient MH profiles resemble those associated with C-NHEJ deficiency. However, translocation junctions between c-myc DSB and general DSBs genome-wide are not MH-biased in ATM-deficient versus wild-type CH12F3 cells and are less biased in 53BP1- and C-NHEJ−deficient cells than CSR junctions or c-myc to S region translocation junctions. We discuss potential roles of DSBR factors in suppressing increased MH-mediated DSB end-joining and features of S regions that may render their DSBs prone to MH-biased end-joining in the absence of DSBR factors.

scholarly journals Comparison of High- and Low-LET Radiation-Induced DNA Double-Strand Break Processing in Living Cells

2020 ◽  
Vol 21 (18) ◽  
pp. 6602 ◽  
Author(s):  
Stefan J. Roobol ◽  
Irene van den Bent ◽  
Wiggert A. van Cappellen ◽  
Tsion E. Abraham ◽  
Maarten W. Paul ◽  
...  
Keyword(s):  
Dna Damage ◽  
Protein A ◽  
Strand Break ◽  
Double Strand Break ◽  
Protein Accumulation ◽  
Dna Double Strand Breaks ◽  
X Ray ◽  
High Let ◽  
Dna End Resection ◽  
Α Particles

High-linear-energy-transfer (LET) radiation is more lethal than similar doses of low-LET radiation types, probably a result of the condensed energy deposition pattern of high-LET radiation. Here, we compare high-LET α-particle to low-LET X-ray irradiation and monitor double-strand break (DSB) processing. Live-cell microscopy was used to monitor DNA double-strand breaks (DSBs), marked by p53-binding protein 1 (53BP1). In addition, the accumulation of the endogenous 53BP1 and replication protein A (RPA) DSB processing proteins was analyzed by immunofluorescence. In contrast to α-particle-induced 53BP1 foci, X-ray-induced foci were resolved quickly and more dynamically as they showed an increase in 53BP1 protein accumulation and size. In addition, the number of individual 53BP1 and RPA foci was higher after X-ray irradiation, while focus intensity was higher after α-particle irradiation. Interestingly, 53BP1 foci induced by α-particles contained multiple RPA foci, suggesting multiple individual resection events, which was not observed after X-ray irradiation. We conclude that high-LET α-particles cause closely interspaced DSBs leading to high local concentrations of repair proteins. Our results point toward a change in DNA damage processing toward DNA end-resection and homologous recombination, possibly due to the depletion of soluble protein in the nucleoplasm. The combination of closely interspaced DSBs and perturbed DNA damage processing could be an explanation for the increased relative biological effectiveness (RBE) of high-LET α-particles compared to X-ray irradiation.

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