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.

scholarly journals An Oligomerized 53BP1 Tudor Domain Suffices for Recognition of DNA Double-Strand Breaks

2008 ◽  
Vol 29 (4) ◽  
pp. 1050-1058 ◽  
Author(s):  
Omar Zgheib ◽  
Kristopher Pataky ◽  
Juergen Brugger ◽  
Thanos D. Halazonetis
Keyword(s):  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Checkpoint Proteins ◽  
Strand Breaks ◽  
Histone H2ax ◽  
Dna Dsbs ◽  
Tudor Domain ◽  
Histone Core ◽  
Histone H2ax Phosphorylation ◽  
Oligomerization Domain

ABSTRACT 53BP1, the vertebrate ortholog of the budding yeast Rad9 and fission yeast Crb2/Rhp9 checkpoint proteins, is recruited rapidly to sites of DNA double-strand breaks (DSBs). A tandem tudor domain in human 53BP1 that recognizes methylated residues in the histone core is necessary, but not sufficient, for efficient recruitment. By analysis of deletion mutants, we identify here additional elements in 53BP1 that facilitate recognition of DNA DSBs. The first element corresponds to an independently folding oligomerization domain. Replacement of this domain with heterologous tetramerization domains preserves the ability of 53BP1 to recognize DNA DSBs. A second element is only about 15 amino acids long and appears to be a C-terminal extension of the tudor domain, rather than an independently functioning domain. Recruitment of 53BP1 to sites of DNA DSBs is facilitated by histone H2AX phosphorylation and ubiquitination. However, none of the 53BP1 domains/elements important for recruitment are known to bind phosphopeptides or ubiquitin, suggesting that histone phosphorylation and ubiquitination regulate 53BP1 recruitment to sites of DNA DSBs indirectly.

scholarly journals Phosphorylation of BLM, Dissociation from Topoisomerase IIIα, and Colocalization with γ-H2AX after Topoisomerase I-Induced Replication Damage

2005 ◽  
Vol 25 (20) ◽  
pp. 8925-8937 ◽  
Author(s):  
V. Ashutosh Rao ◽  
Angela M. Fan ◽  
LingHua Meng ◽  
Christopher F. Doe ◽  
Phillip S. North ◽  
...  
Keyword(s):  
Topoisomerase I ◽  
Protein A ◽  
Dna Helicase ◽  
Double Strand Breaks ◽  
Nuclear Bodies ◽  
H2ax Phosphorylation ◽  
Strand Breaks ◽  
Histone H2ax ◽  
Pml Nuclear Bodies ◽  
Atm Protein

ABSTRACT Topoisomerase I-associated DNA single-strand breaks selectively trapped by camptothecins are lethal after being converted to double-strand breaks by replication fork collisions. BLM (Bloom's syndrome protein), a RecQ DNA helicase, and topoisomerase IIIα (Top3α) appear essential for the resolution of stalled replication forks (Holliday junctions). We investigated the involvement of BLM in the signaling response to Top1-mediated replication DNA damage. In BLM-complemented cells, BLM colocalized with promyelocytic leukemia protein (PML) nuclear bodies and Top3α. Fibroblasts without BLM showed an increased sensitivity to camptothecin, enhanced formation of Top1-DNA complexes, and delayed histone H2AX phosphorylation (γ-H2AX). Camptothecin also induced nuclear relocalization of BLM, Top3α, and PML protein and replication-dependent phosphorylation of BLM on threonine 99 (T99p-BLM). T99p-BLM was also observed following replication stress induced by hydroxyurea. Ataxia telangiectasia mutated (ATM) protein and AT- and Rad9-related protein kinases, but not DNA-dependent protein kinase, appeared to play a redundant role in phosphorylating BLM. Following camptothecin treatment, T99p-BLM colocalized with γ-H2AX but not with Top3α or PML. Thus, BLM appears to dissociate from Top3α and PML following its phosphorylation and facilitates H2AX phosphorylation in response to replication double-strand breaks induced by Top1. A defect in γ-H2AX signaling in response to unrepaired replication-mediated double-strand breaks might, at least in part, explain the camptothecin-sensitivity of BLM-deficient cells.

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.

Sign in / Sign up

Export Citation Format

Share Document