scholarly journals Dynamic localization of SPO11-1 and conformational changes of meiotic axial elements during recombination initiation of maize meiosis

2018 ◽  
Author(s):  
Jia-Chi Ku ◽  
Arnaud Ronceret ◽  
Inna Golubovskaya ◽  
Ding Hua Lee ◽  
Chiting Wang ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Super Resolution ◽  
Double Strand Breaks ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Dynamic Localization ◽  
Strand Breaks ◽  
Meiotic Chromosomes ◽  
Related Enzyme

AbstractMeiotic double-strand breaks (DSBs) are generated by the evolutionarily conserved SPO11 complex in the context of chromatin loops that are organized along axial elements (AEs) of chromosomes. However, how DSBs are formed with respect to chromosome axes and the SPO11 complex remains unclear in plants. Here, we confirm that DSB and bivalent formation are defective in maize spo11-1 mutants. Super-resolution microscopy demonstrates dynamic localization of SPO11-1 during recombination initiation, with variable numbers of SPO11-1 foci being distributed in nuclei but similar numbers of SPO11-1 foci being found on AEs. Notably, cytological analysis of spo11-1 meiocytes revealed an aberrant AE structure. At leptotene, AEs of wild-type and spo11-1 meiocytes were similarly curly and discontinuous. However, during early zygotene, wild-type AEs become uniform and exhibit shortened axes, whereas the elongated and curly AEs persisted in spo11-1 mutants, suggesting that loss of SPO11-1 compromised AE structural maturation. Our results reveal an interesting relationship between SPO11-1 loading onto AEs and the conformational remodeling of AEs during recombination initiation.Author SummaryMeiosis is essential during sexual reproduction to produce haploid gametes. Recombination is the most crucial step during meiotic prophase I. It enables pairing of homologous chromosomes prior to their reductional division and generates new combinations of genetic alleles for transmission to the next generation. Meiotic recombination is initiated by generating DNA double-strand breaks (DSBs) via SPO11, a topoisomerase-related enzyme. The activity, timing and location of this DSB machinery must be controlled precisely, but how this is achieved remains obscure. Here, we show dynamic localization of SPO11-1 on chromatin during meiotic initiation in maize, yet a similar number of SPO11-1 is able to load onto axial elements (AEs), which accompanies a structural change of the AEs of wild-type meiotic chromosomes. Interestingly, loss of SPO11-1 not only affects DSB formation but also impairs structural alterations of AEs, resulting in abnormally long and curly AEs during early meiosis. Our study provides new insights into SPO11-1 localization during recombination initiation and suggests an intimate relationship between DSB formation and AE structural changes.

scholarly journals The Karyopherin Kap142p/Msn5p Mediates Nuclear Import and Nuclear Export of Different Cargo Proteins

2001 ◽  
Vol 152 (4) ◽  
pp. 729-740 ◽  
Author(s):  
Kimihisa Yoshida ◽  
Günter Blobel
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Protein Import ◽  
Protein A ◽  
Double Strand Breaks ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Highly Sensitive ◽  
Cargo Proteins

We have identified a novel pathway for protein import into the nucleus. Although the product of Saccharomyces cerevisiae gene MSN5 was previously shown to function as a karyopherin (Kap) for nuclear export of various proteins, we discovered a nuclear import pathway mediated by Msn5p (also referred to as Kap142p). We have purified from yeast cytosol a complex containing Kap142p and the trimeric replication protein A (RPA), which is required for multiple aspects of DNA metabolism, including DNA replication, DNA repair, and recombination. In wild-type cells, RPA was localized primarily to the nucleus but, in a KAP142 deletion strain, RPA was mislocalized to the cytoplasm and the strain was highly sensitive to bleomycin (BLM). BLM causes DNA double-strand breaks and, in S. cerevisiae, the DNA damage is repaired predominantly by RPA-dependent homologous recombination. Therefore, our results indicate that in wild-type cells a critical portion of RPA was imported into the nucleus by Kap142p. Like several other import-related Kap–substrate complexes, the endogenous RPA–Kap142p complex was dissociated by RanGTP, but not by RanGDP. All three RPA genes are essential for viability, whereas KAP142 is not. Perhaps explaining this disparity, we observed an interaction between RPA and Kap95p in a strain lacking Kap142p. This interaction could provide a mechanism for import of RPA into the nucleus and cell viability in the absence of Kap142p. Together with published results (Kaffman, A., N.M. Rank, E.M. O'Neill, L.S. Huang, and E.K. O'Shea. 1998. Nature. 396:482–486; Blondel, M., P.M. Alepuz, L.S. Huang, S. Shaham, G. Ammerer, and M. Peter. 1999. Genes Dev. 13:2284–2300; DeVit, M.J., and M. Johnston. 1999. Curr. Biol. 9:1231–1241; Mahanty, S.K., Y. Wang, F.W. Farley, and E.A. Elion. 1999. Cell. 98:501–512) our data indicate that the karyopherin Kap142p is able to mediate nuclear import of one set of proteins and nuclear export of a different set of proteins.

scholarly journals Interleukin33 deficiency causes tau abnormality and neurodegeneration with Alzheimer-like symptoms in aged mice

2017 ◽  
Vol 7 (7) ◽  
pp. e1164-e1164 ◽  
Author(s):  
C Carlock ◽  
J Wu ◽  
J Shim ◽  
I Moreno-Gonzalez ◽  
M R Pitcher ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Memory Impairment ◽  
Middle Age ◽  
Late Onset ◽  
Double Strand Breaks ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Rapid Accumulation ◽  
Do So

Abstract Late-onset Alzheimer’s disease (AD) remains a medical mystery. Recent studies have linked it to impaired repair of aged neurons. Potential involvement of interleukin33 (IL33) in AD has been reported. Here we show that IL33, which was expressed by up to 75% astrocytes in the aged brains, was critical for repair of aged neurons. Mice lacking Il33 gene (Il33 −/− ) developed AD-like disease after 60–80 weeks, which was characterized by tau abnormality and a heavy loss of neurons/neurites in the cerebral cortex and hippocampus accompanied with cognition/memory impairment. We detected an abrupt aging surge in the cortical and hippocampal neurons at middle age (40 weeks). To counter the aging surge, wild-type mice rapidly upregulated repair of DNA double-strand breaks (DSBs) and autophagic clearance of cellular wastes in these neurons. Il33 −/− mice failed to do so, but instead went on to develop rapid accumulation of abnormal tau, massive DSBs and abnormal autophagic vacuoles in these neurons. Thus, uncontrolled neuronal aging surge at middle age due to lack of IL33 resulted in neurodegeneration and late-onset AD-like symptome in Il33 −/− mice. Our study also suggests that the aging surge is a time to search for biomarkers for early diagnosis of AD before massive neuron loss.

scholarly journals Critical Role of RecN in Recombinational DNA Repair and Survival of Helicobacter pylori

2007 ◽  
Vol 76 (1) ◽  
pp. 153-160 ◽  
Author(s):  
Ge Wang ◽  
Robert J. Maier
Keyword(s):  
Helicobacter Pylori ◽  
Parent Strain ◽  
Dna Recombination ◽  
Double Strand Breaks ◽  
Recombinational Repair ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Air Exposure ◽  
Strand Breaks ◽  
H Pylori

ABSTRACT Homologous recombination is one of the key mechanisms responsible for the repair of DNA double-strand breaks. Recombinational repair normally requires a battery of proteins, each with specific DNA recognition, strand transfer, resolution, or other functions. Helicobacter pylori lacks many of the proteins normally involved in the early stage (presynapsis) of recombinational repair, but it has a RecN homologue with an unclear function. A recN mutant strain of H. pylori was shown to be much more sensitive than its parent to mitomycin C, an agent predominantly causing DNA double-strand breaks. The recN strain was unable to survive exposure to either air or acid as well as the parent strain, and air exposure resulted in no viable recN cells recovered after 8 h. In oxidative stress conditions (i.e., air exposure), a recN strain accumulated significantly more damaged (multiply fragmented) DNA than the parent strain. To assess the DNA recombination abilities of strains, their transformation abilities were compared by separately monitoring transformation using H. pylori DNA fragments containing either a site-specific mutation (conferring rifampin resistance) or a large insertion (kanamycin resistance cassette). The transformation frequencies using the two types of DNA donor were 10- and 50-fold lower, respectively, for the recN strain than for the wild type, indicating that RecN plays an important role in facilitating DNA recombination. In two separate mouse colonization experiments, the recN strain colonized most of the stomachs, but the average number of recovered cells was 10-fold less for the mutant than for the parent strain (a statistically significant difference). Complementation of the recN strain by chromosomal insertion of a functional recN gene restored both the recombination frequency and mouse colonization ability to the wild-type levels. Thus, H. pylori RecN, as a component of DNA recombinational repair, plays a significant role in H. pylori survival in vivo.

scholarly journals Nonhomologous end-joining of site-specific but not of radiation-induced DNA double-strand breaks is reduced in the presence of wild-type p53

Oncogene ◽  
2005 ◽  
Vol 24 (10) ◽  
pp. 1663-1672 ◽  
Author(s):  
Jochen Dahm-Daphi ◽  
Petra Hubbe ◽  
Fruzsina Horvath ◽  
Raafat A El-Awady ◽  
Katie E Bouffard ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Site Specific ◽  
Strand Breaks ◽  
Radiation Induced ◽  
Wild Type P53

scholarly journals Distinctive nuclear zone for RAD51-mediated homologous recombinational DNA repair

2021 ◽  
Author(s):  
Yasunori Horikoshi ◽  
Hiroki Shima ◽  
Wataru Kobayashi ◽  
Jiying Sun ◽  
Volker J Schmid ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Nuclear Architecture ◽  
Super Resolution ◽  
Double Strand Breaks ◽  
Genome Integrity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Sister Chromatids ◽  
Super Resolution Microscopy ◽  
Damaged Dna

Genome-based functions are inseparable from the dynamic higher-order architecture of the cell nucleus. In this context, the repair of DNA damage is coordinated by precise spatiotemporal controls that target and regulate the repair machinery required to maintain genome integrity. However, the mechanisms that pair damaged DNA with intact template for repair by homologous recombination (HR) without illegitimate recombination remain unclear. This report highlights the intimate relationship between nuclear architecture and HR in mammalian cells. RAD51, the key recombinase of HR, forms spherical foci in S/G2 phases spontaneously. Using super-resolution microscopy, we show that following induction of DNA double-strand breaks RAD51 foci at damaged sites elongate to bridge between intact and damaged sister chromatids; this assembly occurs within bundle-shaped distinctive nuclear zones, requires interactions of RAD51 with various factors, and precedes ATP-dependent events involved the recombination of intact and damaged DNA. We observed a time-dependent transfer of single-stranded DNA overhangs, generated during HR, into such zones. Our observations suggest that RAD51-mediated homologous pairing during HR takes place within the distinctive nuclear zones to execute appropriate recombination.

Super-resolution localization microscopy of radiation-induced histone H2AX-phosphorylation in relation to H3K9-trimethylation in HeLa cells

Nanoscale ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 4320-4331 ◽  
Author(s):  
Michael Hausmann ◽  
Emma Wagner ◽  
Jin-Ho Lee ◽  
Gerrit Schrock ◽  
Wladimir Schaufler ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Super Resolution ◽  
Double Strand Breaks ◽  
H2ax Phosphorylation ◽  
Strand Breaks ◽  
Localization Microscopy ◽  
Histone H2ax ◽  
H3k9 Trimethylation ◽  
Radiation Induced ◽  
Histone H2ax Phosphorylation

Ionizing radiation (IR)-induced damage confers functional and conformational changes to nuclear chromatin associated with DNA single and double strand breaks.

Cytosolic Histone H1 Releasing under Different Apoptotic Stimuli in Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4790-4790
Author(s):  
Eva Gine ◽  
Marta Crespo ◽  
Emili Montserrat ◽  
Francesc Bosch
Keyword(s):  
Functional Status ◽  
Peripheral Blood ◽  
Histone H1 ◽  
Double Strand Breaks ◽  
Fish Analysis ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cells In Culture

Abstract Recently, it has been demonstrated that nuclear histone H1 could be released into cytoplasm when apoptosis is induced by DNA double-strand breaks, this process being dependent of p53 functional status. In addition, cytosolic histone H1.2 was shown to induce cytochrome C release in a Bak-dependent manner. The aims of this study were: 1) to analyze the presence of histone H1.2 in the cytosol of peripheral blood purified CLL cells during apoptosis induced by different chemotherapeutic drugs or ionizing radiation in CLL cells in culture; 2) to correlate the presence of cytosolic histone H1 with the p53 functional status. FISH analysis was used to select samples with (n=2) or without (n=4) p53 deletion. Peripheral blood purified CLL cells were cultured for 24 hours with no treatment, after irradiation (25 Gy), in the presence of fludarabine and mitoxantrone (FM) (1mg/ml and 0.5 mg/ml respectively), or with etoposide (5.8 mg/ml). Cell viability and analysis of apoptosis were performed by annexin V/PI staining and FACscan analysis. Cytosolic and nuclear fractions were separated with the Nuclear/Cytosol Fractionation kit (Biovision). The presence of histone H1 in the cytosolic fraction was assessed by Western Blott using the anti-histone H1 antibody (Upstate). Nuclear contamination of this fraction was detected by using anti-ribonucleoprotein (hnRNP) antibody (Abcam). In p53 wild-type CLL cases, increased apoptosis was observed under all stimuli, being the FM combination the most effective. In contrast, CLL cases with p53 deletion displayed a lower or no response to the different treatments. In p53 wild-type CLL cases, histone H1 was only observed in cytosolic fractions at 24 hours when irradiation or etoposide treatment was applied, but not with FM treatment. Interestingly, cytosolic histone H1 was also present in the control cells at 24h of culture, indicating that H1 was also involved in the spontaneous apoptosis observed due to the microambient deprivation of CLL cells in culture. This histone H1 traffic was also confirmed using immunofluorescence analysis. Conversely, in p53 deleted CLL cases histone H1 was not detected into cytosolic fractions with any treatment. In conclusion, cytosolic histone H1 appeared in CLL cases in a p53 dependent manner when DNA double-strand breaks were induced. These results suggest that histone H1 plays an important role in the p53-dependent cytosol apoptotic signaling.

scholarly journals Rec8 Guides Canonical Spo11 Distribution along Yeast Meiotic Chromosomes

2009 ◽  
Vol 20 (13) ◽  
pp. 3064-3076 ◽  
Author(s):  
Kazuto Kugou ◽  
Tomoyuki Fukuda ◽  
Shintaro Yamada ◽  
Masaru Ito ◽  
Hiroyuki Sasanuma ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Spatial Regulation ◽  
Tiling Arrays ◽  
Meiotic Chromosomes ◽  
Genome Wide ◽  
Hydroxyurea Treatment ◽  
Temporal And Spatial

Spo11-mediated DNA double-strand breaks (DSBs) that initiate meiotic recombination are temporally and spatially controlled. The meiotic cohesin Rec8 has been implicated in regulating DSB formation, but little is known about the features of their interplay. To elucidate this point, we investigated the genome-wide localization of Spo11 in budding yeast during early meiosis by chromatin immunoprecipitation using high-density tiling arrays. We found that Spo11 is dynamically localized to meiotic chromosomes. Spo11 initially accumulated around centromeres and thereafter localized to arm regions as premeiotic S phase proceeded. During this stage, a substantial proportion of Spo11 bound to Rec8 binding sites. Eventually, some of Spo11 further bound to both DSB and Rec8 sites. We also showed that such a change in a distribution of Spo11 is affected by hydroxyurea treatment. Interestingly, deletion of REC8 influences the localization of Spo11 to centromeres and in some of the intervals of the chromosomal arms. Thus, we observed a lack of DSB formation in a region-specific manner. These observations suggest that Rec8 would prearrange the distribution of Spo11 along chromosomes and will provide clues to understanding temporal and spatial regulation of DSB formation.

scholarly journals RexAB promotes the survival of Staphylococcus aureus exposed to multiple classes of antibiotics

2021 ◽  
Author(s):  
Rebecca S. Clarke ◽  
Kam Pou Ha ◽  
Andrew M. Edwards
Keyword(s):  
Reactive Oxygen Species ◽  
Staphylococcus Aureus ◽  
Dna Damage ◽  
Sos Response ◽  
Double Strand Breaks ◽  
Oxygen Species ◽  
Bacterial Survival ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Antibiotics inhibit essential bacterial processes, resulting in arrest of growth and in some cases cell death. Many antibiotics are also reported to trigger endogenous production of reactive oxygen species (ROS), which damage DNA, leading to induction of the mutagenic SOS response associated with the emergence of drug resistance. However, the type of DNA damage that arises and how this triggers the SOS response is largely unclear. We found that several different classes of antibiotic triggered dose-dependent induction of the SOS response in Staphylococcus aureus , indicative of DNA damage, including some bacteriostatic drugs. The SOS response was heterogenous and varied in magnitude between strains and antibiotics. However, in many cases, full induction of the SOS response was dependent upon the RexAB helicase/nuclease complex, which processes DNA double strand breaks to produce single-stranded DNA and facilitate RecA nucleoprotein filament formation. The importance of RexAB in repair of DNA was confirmed by measuring bacterial survival during antibiotic exposure, with most drugs having significantly greater bactericidal activity against rexB mutants relative to wild type strains. For some, but not all antibiotics there was no difference in bactericidal activity between wild type and rexB mutant under anaerobic conditions, indicative of a role for reactive oxygen species in mediating DNA damage. Taken together, this work confirms previous observations that several classes of antibiotics cause DNA damage in S. aureus and extends them by showing that processing of DNA double strand breaks by RexAB is a major trigger of the mutagenic SOS response and promotes bacterial survival.

scholarly journals Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors

Genes ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 511 ◽  
Author(s):  
Duran Sürün ◽  
Aksana Schneider ◽  
Jovan Mircetic ◽  
Katrin Neumann ◽  
Felix Lansing ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
High Efficiency ◽  
Double Strand Breaks ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cas9 Nuclease ◽  
Human Ipscs ◽  
Human Ips Cells ◽  
Induced Pluripotent

In contrast to CRISPR/Cas9 nucleases, CRISPR base editors (BE) and prime editors (PE) enable predefined nucleotide exchanges in genomic sequences without generating DNA double strand breaks. Here, we employed BE and PE mRNAs in conjunction with chemically synthesized sgRNAs and pegRNAs for efficient editing of human induced pluripotent stem cells (iPSC). Whereas we were unable to correct a disease-causing mutation in patient derived iPSCs using a CRISPR/Cas9 nuclease approach, we corrected the mutation back to wild type with high efficiency utilizing an adenine BE. We also used adenine and cytosine BEs to introduce nine different cancer associated TP53 mutations into human iPSCs with up to 90% efficiency, generating a panel of cell lines to investigate the biology of these mutations in an isogenic background. Finally, we pioneered the use of prime editing in human iPSCs, opening this important cell type for the precise modification of nucleotides not addressable by BEs and to multiple nucleotide exchanges. These approaches eliminate the necessity of deriving disease specific iPSCs from human donors and allows the comparison of different disease-causing mutations in isogenic genetic backgrounds.

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